Multispecific antibodies

ABSTRACT

The invention provides multispecific antibodies and methods of making and using such antibodies.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.13/030,760, filed Feb. 18, 2011, which is a continuation of U.S. patentapplication Ser. No. 12/552,177, filed Sep. 1, 2009, now U.S. Pat. No.8,193,321, which claims benefit from U.S. Provisional Application No.61/190,856, filed Sep. 3, 2008, each of which is herein incorporated byreference.

REFERENCE TO A COMPUTER PROGRAM LISTING APPENDIX

A Sequence Listing is provided in this patent document as a txt file.The content of this file is herein incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to multispecific antibodies, and methodsof making and using such antibodies.

BACKGROUND OF THE INVENTION

Antibodies are specific immunoglobulin polypeptides produced by thevertebrate immune system in response to challenge by foreign proteins,glycoproteins, cells, or other antigenic foreign substances. Animportant part of this process is the generation of antibodies that bindspecifically to a particular foreign substance. The binding specificityof such polypeptides to a particular antigen is highly refined, and themultitude of specificities capable of being generated by the individualvertebrate is remarkable in its complexity and variability. Thousands ofantigens are capable of eliciting responses, each almost exclusivelydirected to the particular antigen which elicited it.

Specific antigen recognition is essential for antibodies to function inthe adaptive immune response. The combinatorial association of heavychain (HC) and light chain (LC) is conserved in all vertebrates in thegeneration of the antibody repertoire. There is, however, asymmetry ofdiversity in the two chains. The variable domain of HC (V_(H)) containssignificantly higher sequence diversity and contributes the determinantsof antigen recognition more often than the variable domain of the LC(V_(L)). The role of the LC in determining antigen-specificity isindicated by a process called receptor editing. Ongoing recombination ofthe V_(L) genes to edit the B cell receptor is the main mechanism tocorrect self reactive antibody precursors, which appear to constitute asignificant portion of the initial repertoire (˜75%). Altering of thelight chain is demonstrated to extinguish unwanted binding specificityor multi-specificity.

The specificity of antibodies and antibody fragments for a particularantigen or antigens makes antibodies desirable therapeutic agents.Antibodies and antibody fragments can be used to target particulartissues, for example, a tumor, and thereby minimize the potential sideeffects of non-specific targeting. As such, there is a current andcontinuing need to identify and characterize therapeutic antibodies,especially antibodies, fragments, and derivatives thereof, useful in thetreatment of cancer and other proliferative disorders.

SUMMARY OF THE INVENTION

The present invention provides an isolated antibody containing ahypervariable region (HVR) L1 sequence containing the sequence NIAKTISGY(SEQ ID NO:1), where the antibody specifically binds human epidermalgrowth factor receptor 2 (HER2) and vascular endothelial growth factor(VEGF). In one embodiment, the antibody further contains an HVR-L2containing the sequence WGSFLY (SEQ ID NO: 2) and/or an HVR-L3containing the sequence HYSSPP (SEQ ID NO: 3). In another embodiment,the antibody further contains, one, two, or three HVR sequences selectedfrom (i) HVR-H1 containing the sequence NIKDTY (SEQ ID NO:4); (ii)HVR-H2 containing the sequence RIYPTNGYTR (SEQ ID NO:5); and (iii)HVR-H3 containing the sequence WGGDGFYAMD (SEQ ID NO:6). In anotherembodiment, the antibody further contains, one, two, or three HVRsequences selected from (i) HVR-H1 containing the sequence NISGTY (SEQID NO:7); (ii) HVR-H2 containing the sequence RIYPSEGYTR (SEQ ID NO:8);and (iii) HVR-H3 containing the sequence WVGVGFYAMD (SEQ ID NO:9).

In another aspect, the invention features an isolated antibodycontaining an HVR-L1 sequence containing the sequence X₁IX₃X₄X₅X₆X₇X₈X₉Y (SEQ ID NO: 83), wherein X₁ is any amino acid exceptaspartic acid, X₃ is any amino acid except proline, X₄ is any amino acidexcept arginine, and X₅ is any amino acid except serine, where theantibody specifically binds HER2 and VEGF. In one embodiment, anantibody containing the sequence X₁I X₃X₄X₅X₆X₇X₈X₉Y (SEQ ID NO: 83) hasan asparagine at X₁, an alanine at X₃, a lysine at X₄, a threonine atX₅, a serine at X₇, and/or a glycine at X₈, or any combination thereof.In various embodiments of this aspect of the invention, any of theHVR-L1 residues shown in FIG. 57 to have an F value of greater than 1,5, or 10 are residues that are preferably maintained as the same residuefound in the same position of the HVR-L1 of bH1-44 or bH1-81 (SEQ ID NO:1). In additional embodiments, any of the HVR-L1 residues shown in Table14 to have ΔΔG values greater than 1 are residues that are preferablymaintained as the same residue found in the same position of the HVR-L1of bH1-44 or bH1-81 (SEQ ID NO: 1). In one embodiment, the antibodycomprises an HVR-H2 sequence comprising the sequence RX₂X₃X₄X₅X₆X₇X₈X₉R(SEQ ID NO: 84). In one embodiment, the antibody further contains anHVR-L2 containing the sequence WGSFLY (SEQ ID NO: 2) and/or an HVR-L3containing the sequence HYSSPP (SEQ ID NO: 3). In another embodiment,the antibody further contains, one, two, or three HVR sequences selectedfrom (i) HVR-H1 containing the sequence NIKDTY (SEQ ID NO:4); (ii)HVR-H2 containing the sequence RIYPTNGYTR (SEQ ID NO:5); and (iii)HVR-H3 containing the sequence WGGDGFYAMD (SEQ ID NO:6). In anotherembodiment, the antibody further contains, one, two, or three HVRsequences selected from (i) HVR-H1 containing the sequence NISGTY (SEQID NO:7); (ii) HVR-H2 containing the sequence RIYPSEGYTR (SEQ ID NO:8);and (iii) HVR-H3 containing the sequence WVGVGFYAMD (SEQ ID NO:9).

In another aspect, the invention features an isolated antibodycontaining an HVR-H2 sequence containing the sequence RX₂X₃X₄X₅X₆X₇X₈X₉R(SEQ ID NO: 85), wherein X₅ is any amino acid except threonine and X₆ isany amino acid except asparagine and where the antibody specificallybinds HER2 and VEGF. In another embodiment, an antibody containing thesequence RX₂X₃ X₄X₅X₆X₇X₈X₉R (SEQ ID NO: 84) has a tyrosine at X₈. Inone embodiment, an antibody containing the sequence RX₂X₃X₄X₅X₆X₇X₈X₉R(SEQ ID NO: 84) has a serine at X₅ and/or a glutamic acid at X₆. Inanother embodiment of this aspect, the antibodies further contain one,two, or three HVR sequences selected from the group of a HVR-L1containing the sequence NIAKTISGY (SEQ ID NO: 1), a HVR-L2 containingthe sequence WGSFLY (SEQ ID NO: 2), and/or a HVR-L3 containing thesequence HYSSPP (SEQ ID NO: 3). In any of the embodiments describedherein, the antibodies further contain, one or two HVR sequencesselected from (i) HVR-H1 containing the sequence NIKDTY (SEQ ID NO:4)and (ii) HVR-H3 containing the sequence WGGDGFYAMD (SEQ ID NO:6). In anadditional embodiment, the antibodies further contain, one or two HVRsequences selected from (i) HVR-H1 containing the sequence NISGTY (SEQID NO:7) and (ii) HVR-H3 containing the sequence WVGVGFYAMD (SEQ IDNO:9).

In various embodiments of this aspect of the invention, any of theHVR-H2 residues shown in FIG. 57 to have an F value of greater than 1,5, or 10 are residues that are preferably maintained as the same residuefound in the same position of the HVR-H2 of bH1-44 or bH1-81 (SEQ IDNOS: 8 and 5, respectively). In additional embodiments, any of theHVR-H2 residues shown in Table 14 to have ΔΔG values greater than 1 areresidues that are preferably maintained as the same residue found in thesame position of the HVR-H2 of bH1-44 or bH1-81 (SEQ ID NOS: 8 and 5,respectively).

In particular embodiments, the antibody contains an HVR-L1 sequencecontaining NIAKTISGY (SEQ ID NO:1); an HVR-L2 sequence containing WGSFLY(SEQ ID NO:2); an HVR-L3 sequence containing HYSSPP (SEQ ID NO:3); anHVR-H1 sequence containing NIKDTY (SEQ ID NO:4); an HVR-H2 sequencecontaining RIYPTNGYTR (SEQ ID NO:5); and an HVR-H3 sequence containingWGGDGFYAMD (SEQ ID NO:6) or contains an HVR-L1 sequence containingNIAKTISGY (SEQ ID NO:1); an HVR-L2 sequence containing WGSFLY (SEQ IDNO:2); an HVR-L3 sequence containing HYSSPP (SEQ ID NO:3); an HVR-H1sequence containing NISGTY (SEQ ID NO:7); an HVR-H2 sequence containingRIYPSEGYTR (SEQ ID NO: 8); and/or an HVR-H3 sequence containingWVGVGFYAMD (SEQ ID NO:9).

In a further particular embodiment the isolated antibody containsHVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2, and HVR-H3, wherein each, inorder, contains the sequence NIAKTISGY (SEQ ID NO:1); WGSFLY (SEQ IDNO:2); HYSSPP (SEQ ID NO:3); NIKDTY (SEQ ID NO:4); RIYPTNGYTR (SEQ IDNO:5); and WGGDGFYAMD (SEQ ID NO:6) and specifically binds HER2 andVEGF. In another particular embodiment, the antibody contains HVR-L1,HVR-L2, HVR-L3, HVR-H1, HVR-H2, and HVR-H3, wherein each, in order,contains the sequence NIAKTISGY (SEQ ID NO:1); WGSFLY (SEQ ID NO:2);HYSSPP (SEQ ID NO:3); NISGTY (SEQ ID NO:7); RIYPSEGYTR (SEQ ID NO:8);and WVGVGFYAMD (SEQ ID NO:9) and specifically binds HER2 and VEGF.

In various embodiments of any of the aspects described herein, theantibody binds human and murine VEGF with a Kd of 150 nM or stronger andHER2 with a Kd of 7 nM or stronger. In additional embodiments, theantibody inhibits VEGF-induced cell proliferation and proliferation of aHER2 expressing cell relative to a control. In a particular embodiment,the antibody binds human and murine VEGF with a Kd of 36 nM or strongerand HER2 with a Kd of 1 nM or stronger. In an additional embodiment, theantibody inhibits VEGF binding to VEGFR2.

In another aspect, the invention features an isolated antibody thatbinds human and murine VEGF with a Kd of 150 nM or stronger and HER2with a Kd of 7 nM or stronger and wherein the antibody inhibitsVEGF-induced cell proliferation and proliferation of a HER2 expressingcell relative to a control. In one embodiment, the antibody binds humanand murine VEGF with a Kd of 36 nM or stronger and HER2 with a Kd of 1nM or stronger.

In yet another aspect the invention provides an isolated antibodyfragment that binds human VEGF with a Kd of 58 nM or stronger and HER2with a Kd of 6 nM or stronger, and/or inhibits VEGF-induced cellproliferation and proliferation of a HER2 expressing cell relative to acontrol. In a particular embodiment, the antibody fragment binds humanand murine VEGF with a Kd of 33 nM or stronger and HER2 with a Kd of 0.7nM or stronger. In another particular embodiment, the fragment is a Fabfragment or a single chain variable fragment (scFv).

In any of the above-described aspects, the antibody may be a monoclonalantibody. In another embodiment of all the above aspects, the antibodymay be an IgG antibody. In additional embodiments of all the aboveaspects, at least a portion of the framework sequence of the antibodymay be a human consensus framework sequence.

In another aspect, the invention features a fragment of an antibody anyof the antibodies described herein. One embodiment of an antibodyfragment is a fragment containing a HVR-L1 sequence containing thesequence NIAKTISGY (SEQ ID NO:1) that specifically binds HER2 and VEGF.In another embodiment, the antibody fragment further contains one or twoHVR sequences selected from (i) HVR-L2 containing the sequence WGSFLY(SEQ ID NO:2); and (ii) HVR-L3 containing the sequence HYSSPP (SEQ IDNO:3). In another embodiment, the antibody fragment further contains,one, two, or three HVR sequences selected from (i) HVR-H1 containing thesequence NIKDTY (SEQ ID NO:4); (ii) HVR-H2 containing the sequenceRIYPTNGYTR (SEQ ID NO:5); and (iii) HVR-H3 containing the sequenceWGGDGFYAMD (SEQ ID NO:6). In an additional embodiment, the antibodyfragment further contains, one, two, or three HVR sequences selectedfrom (i) HVR-H1 containing the sequence NISGTY (SEQ ID NO:7); (ii)HVR-H2 containing the sequence RIYPSEGYTR (SEQ ID NO:8); and (iii)HVR-H3 containing the sequence WVGVGFYAMD (SEQ ID NO:9). In particularembodiments, the antibody fragment contains an HVR-L1 sequencecontaining NIAKTISGY (SEQ ID NO:1); an HVR-L2 sequence containing WGSFLY(SEQ ID NO:2); an HVR-L3 sequence containing HYSSPP (SEQ ID NO:3); anHVR-H1 sequence containing NIKDTY (SEQ ID NO:4); an HVR-H2 sequencecontaining RIYPTNGYTR (SEQ ID NO:5); and an HVR-H3 sequence containingWGGDGFYAMD (SEQ ID NO:6) or contains an HVR-L1 sequence containingNIAKTISGY (SEQ ID NO:1); an HVR-L2 sequence containing WGSFLY (SEQ IDNO:2); an HVR-L3 sequence containing HYSSPP (SEQ ID NO:3); an HVR-H1sequence containing NISGTY (SEQ ID NO:7); an HVR-H2 sequence containingRIYPSEGYTR (SEQ ID NO:8); and an HVR-H3 sequence containing WVGVGFYAMD(SEQ ID NO:9). In one embodiment, the fragment is a Fab fragment or asingle chain variable fragment (scFv). In additional embodiments of allthe above aspects, at least a portion of the framework sequence of theantibody may be a human consensus framework sequence.

In further aspects, the invention features polynucleotides encoding anyantibody or antibody fragment described herein, as well as a vectorcontaining such a polynucleotide. In particular embodiments, the encodedantibody contains an HVR-L1 sequence containing NIAKTISGY (SEQ ID NO:1).Optionally or additionally, the polynucleotide encodes an antibody thatalso contains an HVR-L2 sequence containing WGSFLY (SEQ ID NO:2); and/oran HVR-L3 sequence containing HYSSPP (SEQ ID NO:3), or any combinationthereof. In an additional aspect, the polynucleotide may further encodean antibody containing one, two, or three of an HVR-H1 sequencecontaining NIKDTY (SEQ ID NO:4); an HVR-H2 sequence containingRIYPTNGYTR (SEQ ID NO:5); and an HVR-H3 sequence containing WGGDGFYAMD(SEQ ID NO:6); or an antibody containing one, two, or three of an HVR-H1containing NISGTY (SEQ ID NO:7); an HVR-H2 containing RIYPSEGYTR (SEQ IDNO:8); and/or an HVR-H3 sequence containing WVGVGFYAMD (SEQ ID NO:9).

In additional aspects of the invention, the polynucleotide encodes anHVR-H1 sequence containing the sequence of NISGTY (SEQ ID NO: 7), anHVR-H2 sequence of RIYPSEGYTR (SEQ ID NO: 8), or an HVR-H3 sequence ofWVGVGFYAMD (SEQ ID NO: 9), or any combination thereof.

In other aspects, the invention features an isolated polynucleotideencoding an HVR-L1 sequence containing the sequence NIAKTISGY (SEQ IDNO:1) and, optionally, the polynucleotide further encodes one, two, orthree HVR sequences selected from (i) an HVR-H1 containing the sequenceNIKDTY (SEQ ID NO:4); (ii) an HVR-H2 containing the sequence RIYPTNGYTR(SEQ ID NO:5); and (iii) an HVR-H3 containing the sequence WGGDGFYAMD(SEQ ID NO:6). In additional aspects, the invention features an isolatedpolynucleotide encoding an HVR-L1 sequence containing the sequenceNIAKTISGY (SEQ ID NO:1); and (i) an HVR-L2 sequence containing thesequence WGSFLY (SEQ ID NO:2) or (ii) an HVR-L3 sequence containing thesequence HYSSPP (SEQ ID NO:3), or both, and, optionally, thepolynucleotide further encodes one, two, or three HVR sequences selectedfrom (i) an HVR-H1 containing the sequence NIKDTY (SEQ ID NO:4); (ii) anHVR-H2 containing the sequence RIYPTNGYTR (SEQ ID NO:5); and (iii) anHVR-H3 containing the sequence WGGDGFYAMD (SEQ ID NO:6).

In a further aspect, the invention features an isolated polynucleotideencoding an HVR-L1 sequence containing the sequence NIAKTISGY (SEQ IDNO:1); an HVR-H1 containing the sequence NISGTY (SEQ ID NO:7); an HVR-H2containing the sequence RIYPSEGYTR (SEQ ID NO:8); and an HVR-H3containing the sequence WVGVGFYAMD (SEQ ID NO:9). In yet another aspect,the invention features an isolated polynucleotide encoding an HVR-L1sequence containing the sequence NIAKTISGY (SEQ ID NO:1); an HVR-L2sequence containing the sequence WGSFLY (SEQ ID NO:2); an HVR-L3sequence containing the sequence HYSSPP (SEQ ID NO:3); an HVR-H1containing the sequence NISGTY (SEQ ID NO:7); an HVR-H2 containing thesequence RIYPSEGYTR (SEQ ID NO:8); and an HVR-H3 containing the sequenceWVGVGFYAMD (SEQ ID NO:9).

In other aspects, the invention features an isolated polynucleotideencoding an HVR-H1 sequence containing the sequence NISGTY (SEQ IDNO:7), an isolated polynucleotide encoding an HVR-H2 sequence containingthe sequence RIYPSEGYTR (SEQ ID NO:8), and an isolated polynucleotideencoding an HVR-H3 sequence containing the sequence WVGVGFYAMD (SEQ IDNO:9). In another aspect, the invention features an isolatedpolynucleotide encoding an polypeptide containing an HVR-H1 sequencecontaining the sequence NISGTY (SEQ ID NO:7); an HVR-H2 sequencecontaining the sequence RIYPSEGYTR (SEQ ID NO: 8); and an HVR-H3sequence containing the sequence WVGVGFYAMD (SEQ ID NO:9).

In an additional embodiment of the invention, the isolatedpolynucleotide encodes an HVR-L1 sequence containing the sequence X₁IX₃X₄X₅X₆X₇X₈X₉Y (SEQ ID NO: 83), wherein X₁ is any amino acid exceptaspartic acid, X₃ is any amino acid except proline, X₄ is any amino acidexcept arginine, and X₅ is any amino acid except serine. In anotherembodiment of the invention, the polynucleotide encodes an HVR-L1sequence containing the sequence X₁I X₃X₄X₅X₆X₇X₈X₉Y (SEQ ID NO: 83),wherein X₁ is any amino acid except Asp, X₃ is any amino acid exceptproline, X₄ is any amino acid except arginine, and X₅ is any amino acidexcept serine; and a HVR-L2 sequence containing the sequence WGSFLY (SEQID NO: 2) and/or an HVR-L3 sequence containing the sequence HYSSPP (SEQID NO: 3). In additional embodiments of this aspect of the invention,the polynucleotide encodes an antibody containing the sequence X₁IX₃X₄X₅X₆X₇X₈X₉Y (SEQ ID NO: 83) that has an asparagine at X_(1,) analanine at X_(3,) a lysine at X_(4,) a threonine at X_(5,) a serine atX_(7,) and/or a glycine at X_(8,) or any combination thereof. In variousembodiments of this aspect of the invention, any of the HVR-L1 residuesshown in FIG. 57 to have an F value of greater than 1, 5, or 10 areresidues that are preferably maintained as the same residue found in thesame position of the HVR-L1 of bH1-44 or bH1-81 (SEQ ID NO: 1). Inadditional embodiments, any of the HVR-L1 residues shown in Table 14 tohave ΔΔG values greater than 1 are residues that are preferablymaintained as the same residue found in the same position of the HVR-L1of bH1-44 or bH1-81 (SEQ ID NO: 1).

In an additional embodiment of the invention, the polynucleotide encodesan HVR-H2 sequence containing the sequence RX₂X₃X₄X₅X₆X₇X₈X₉R (SEQ IDNO: 85), wherein X₅ is any amino acid except threonine and X₆ is anyamino acid except asparagine. In another aspect, the invention providesa polynucleotide encoding an HVR-H1 sequence containing the sequenceNISGTY (SEQ ID NO: 7); an HVR-H2 sequence containing the sequenceRX₂X₃X₄X₅X₆X₇X₈X₉R (SEQ ID NO: 85), wherein X₅ is any amino acid exceptthreonine and X₆ is any amino acid except asparagine; and an HVR-H3sequence containing the sequence WVGVGFYAMD (SEQ ID NO: 9). In anadditional embodiments of the invention, the polynucleotide encodes anHVR-H2 sequence containing the sequence RX₂X₃X₄X₅X₆X₇X₈X₉R (SEQ ID NO:84) that has a serine at X₅, a glutamic acid at X₆, and/or a tyrosine atX₈, or any combination thereof. In various embodiments of this aspect ofthe invention, any of the HVR-H2 residues shown in FIG. 57 to have an Fvalue of greater than 1, 5, or 10 are residues that are preferablymaintained as the same residue found in the same position of the HVR-H2of bH1-44 or bH1-81 (SEQ ID NOS: 8 and 5, respectively). In additionalembodiments, any of the HVR-H2 residues shown in Table 14 to have ΔΔGvalues greater than 1 are residues that are preferably maintained as thesame residue found in the same position of the HVR-H2 of bH1-44 orbH1-81 (SEQ ID NOS: 8 and 5, respectively).

In further aspects, the invention features an isolated polypeptidecontaining an HVR-L1 sequence containing the sequence NIAKTISGY (SEQ IDNO:1) or an isolated polypeptide containing an HVR-L1 sequencecontaining the sequence NIAKTISGY (SEQ ID NO:1); an HVR-L2 sequencecontaining the sequence WGSFLY (SEQ ID NO:2); and/or an HVR-L3 sequencecontaining the sequence HYSSPP (SEQ ID NO:3). In another aspect, theinvention provides a polypeptide containing an HVR-L1 sequencecontaining the sequence X₁IX₃X₄X₅X₆X₇X₈X₉Y (SEQ ID NO: 83), wherein X₁is any amino acid except aspartic acid, X₃ is any amino acid exceptproline, X₄ is any amino acid except arginine, and X₅ is any amino acidexcept serine. In another embodiment of this aspect, the polypeptidecontains the HVR-L1 sequence X₁I X₃X₄X₅X₆X₇X₈X₉Y (SEQ ID NO: 83),wherein X₁ is any amino acid except aspartic acid, X₃ is any amino acidexcept proline, X₄ is any amino acid except arginine, and X₅ is anyamino acid except serine. Optionally, the polypeptide further includesan HVR-L2 sequence containing the sequence WGSFLY (SEQ ID NO: 2) and/oran HVR-L3 sequence containing the sequence HYSSPP (SEQ ID NO: 3). Inparticular embodiments of any of the above aspects that include apolypeptide that contains the sequence X₁IX₃X₄X₅X₆X₇X₈X₉Y (SEQ ID NO:83), there is an asparagine at X_(1,) an alanine at X_(3,) a lysine atX_(4,) a threonine at X_(5,) a serine at X_(7,) and/or a glycine atX_(8,) or any combination thereof. In various embodiments of this aspectof the invention, any of the HVR-L1 residues shown in FIG. 57 to have anF value of greater than 1, 5, or 10 are residues that are preferablymaintained as the same residue found in the same position of the HVR-L1of bH1-44 or bH1-81 (SEQ ID NO: 1). In additional embodiments, any ofthe HVR-L1 residues shown in Table 14 to have ΔΔG values greater than 1are residues that are preferably maintained as the same residue found inthe same position of the HVR-L1 of bH1-44 or bH1-81 (SEQ ID NO: 1).

The invention also provides a polypeptide containing an HVR-H2 sequencecontaining the sequence RX₂X₃X₄X₅X₆X₇X₈X₉R (SEQ ID NO: 85), wherein X₅is any amino acid except threonine and X₆ is any amino acid exceptasparagine. In another aspect of the invention, the polypeptide containsthe HVR-H2 sequence RX₂X₃X₄X₅X₆X₇X₈X₉R (SEQ ID NO: 85), wherein X₅ isany amino acid except threonine and X₆ is any amino acid exceptasparagine, a HVR-H1 sequence containing the sequence NISGTY (SEQ ID NO:7), and an HVR-H3 sequence containing the sequence WVGVGFYAMD (SEQ IDNO: 9). In different embodiments of the above aspects, the polypeptidecontaining the HVR-H2 sequence containing the sequenceRX₂X₃X₄X₅X₆X₇X₈X₉R (SEQ ID NO: 84) has a serine at X₅, a glutamic acidat X₆, and/or a tyrosine at X_(8,) or any combination thereof. Invarious embodiments of this aspect of the invention, any of the HVR-H2residues shown in FIG. 57 to have an F value of greater than 1, 5, or 10are residues that are preferably maintained as the same residue found inthe same position of the HVR-H2 of bH1-44 or bH1-81 (SEQ ID NOS: 8 and5, respectively). In additional embodiments, any of the HVR-H2 residuesshown in Table 14 to have ΔΔG values greater than 1 are residues thatare preferably maintained as the same residue found in the same positionof the HVR-H2 of bH1-44 or bH1-81 (SEQ ID NOS: 8 and 5, respectively).

The invention also provides a polypeptide containing one, two, or threeof an HVR-H1 sequence containing the sequence NISGTY (SEQ ID NO: 7), aHVR-H2 sequence containing the sequence RIYPSEGYTR (SEQ ID NO: 8),and/or an HVR-H3 sequence containing the sequence WVGVGFYAMD (SEQ ID NO:9), or any combination thereof.

In any of the above aspects, the isolated polypeptide may furthercontain one, two, or three of an HVR-L1 sequence containing the sequenceNIAKTISGY (SEQ ID NO:1); an HVR-H1 containing the sequence NIKDTY (SEQID NO:4); an HVR-H2 containing the sequence RIYPTNGYTR (SEQ ID NO:5);and/or an HVR-H3 containing the sequence WGGDGFYAMD (SEQ ID NO:6), orany combination thereof.

In any of the above aspects, the isolated polypeptide may furthercontain one, two, or three of an HVR-L1 sequence containing the sequenceNIAKTISGY (SEQ ID NO:1); an HVR-L2 sequence containing the sequenceWGSFLY (SEQ ID NO:2); an HVR-L3 sequence containing the sequence HYSSPP(SEQ ID NO:3).

In any of the above aspects, the isolated polypeptide may furthercontain an HVR-H1 containing the sequence NIKDTY (SEQ ID NO:4); anHVR-H2 containing the sequence RIYPTNGYTR (SEQ ID NO:5); and/or anHVR-H3 containing the sequence WGGDGFYAMD (SEQ ID NO:6), or anycombination thereof.

In any of the above aspects, the isolated polypeptide may furthercontain one, two, or three HVR sequences selected from an HVR-H1containing the sequence NISGTY (SEQ ID NO: 7); an HVR-H2 sequencecontaining the sequence RIYPSEGYTR (SEQ ID NO: 8); and/or an HVR-H3containing the sequence WVGVGFYAMD (SEQ ID NO: 9), or any combinationthereof.

In additional aspects, the invention features an isolated polypeptidecontaining an HVR-L1 sequence containing the sequence NIAKTISGY (SEQ IDNO:1) and (i) an HVR-L2 sequence containing the sequence WGSFLY (SEQ IDNO:2) or (ii) an HVR-L3 sequence containing the sequence HYSSPP (SEQ IDNO:3), or both; and one, two, of three HVR sequences selected from (i)an HVR-H1 containing the sequence NISGTY (SEQ ID NO:7); (ii) an HVR-H2containing the sequence RIYPSEGYTR (SEQ ID NO:8); and (iii) an HVR-H3containing the sequence WVGVGFYAMD (SEQ ID NO:9).

In additional aspects, the invention features an isolated polypeptidecontaining an HVR-L1 sequence containing the sequence NIAKTISGY (SEQ IDNO:1) and (i) an HVR-L2 sequence containing the sequence WGSFLY (SEQ IDNO:2) or (ii) an HVR-L3 sequence containing the sequence HYSSPP (SEQ IDNO:3), or both; and one, two, of three HVR sequences selected from (i)an HVR-H1 containing the sequence NIKDTY (SEQ ID NO:4); (ii) an HVR-H2containing the sequence RIYPTNGYTR (SEQ ID NO:5); and/or (iii) an HVR-H3containing the sequence WGGDGFYAMD (SEQ ID NO:6), or any combinationthereof.

In further aspects, the invention features an isolated polypeptidecontaining an HVR-H1 sequence containing the sequence NISGTY (SEQ IDNO:7), an isolated polypeptide comprising an HVR-H2 sequence containingthe sequence RIYPSEGYTR (SEQ ID NO:8), and an isolated polypeptidecontaining an HVR-H3 sequence containing the sequence WVGVGFYAMD (SEQ IDNO:9). In yet a further aspect, the invention features an isolatedpolypeptide containing an HVR-H1 sequence containing the sequence NISGTY(SEQ ID NO:7); an HVR-H2 sequence containing the sequence RIYPSEGYTR(SEQ ID NO:8); and an HVR-H3 sequence containing the sequence WVGVGFYAMD(SEQ ID NO:9).

In one embodiment, the invention provides a vector containing any of theabove described polynucleotides of the invention. In another aspect, theinvention features a host cell containing any of the vectors of theinvention. In one embodiment, the host cell is prokaryotic. In anotherembodiment, the host cell is eukaryotic, for example, a mammalian cell.

In another aspect, the invention features a method of producing any ofthe antibodies or antibody fragments described above. This methodincludes culturing a host cell that contains a vector containing apolynucleotide encoding the antibody and recovering the antibody. Incertain embodiments, the polynucleotide encodes an HVR-L1 sequencecontaining the sequence NIAKTISGY (SEQ ID NO:1) and, optionally, thepolynucleotide further encodes an HVR-H1 containing the sequence NIKDTY(SEQ ID NO:4); an HVR-H2 containing the sequence RIYPTNGYTR (SEQ IDNO:5); and an HVR-H3 containing the sequence WGGDGFYAMD (SEQ ID NO:6).In other embodiments, the polynucleotide encodes an HVR-L1 sequencecontaining the sequence NIAKTISGY (SEQ ID NO:1); an HVR-L2 sequencecontaining the sequence WGSFLY (SEQ ID NO:2); and an HVR-L3 sequencecontaining the sequence HYSSPP (SEQ ID NO:3) and, optionally, thepolynucleotide further encodes an HVR-H1 containing the sequence NIKDTY(SEQ ID NO:4); an HVR-H2 containing the sequence RIYPTNGYTR (SEQ IDNO:5); and an HVR-H3 containing the sequence WGGDGFYAMD (SEQ ID NO:6).In another embodiment, the polynucleotide encodes an HVR-L1 sequencecontaining the sequence NIAKTISGY (SEQ ID NO:1); an HVR-H1 containingthe sequence NISGTY (SEQ ID NO:7); an HVR-H2 containing the sequenceRIYPSEGYTR (SEQ ID NO:8); and an HVR-H3 containing the sequenceWVGVGFYAMD (SEQ ID NO:9). In yet another embodiment, the polynucleotideencodes an HVR-L1 sequence containing the sequence NIAKTISGY (SEQ IDNO:1); an HVR-L2 sequence containing the sequence WGSFLY (SEQ ID NO:2);an HVR-L3 sequence containing the sequence HYSSPP (SEQ ID NO:3); anHVR-H1 containing the sequence NISGTY (SEQ ID NO:7); an HVR-H2containing the sequence RIYPSEGYTR (SEQ ID NO:8); and an HVR-H3containing the sequence WVGVGFYAMD (SEQ ID NO:9).

In further embodiments, the polynucleotide encodes an HVR-H1 sequencecontaining the sequence NISGTY (SEQ ID NO:7), an HVR-H2 sequencecontaining the sequence RIYPSEGYTR (SEQ ID NO: 8), or an HVR-H3 sequencecontaining the sequence WVGVGFYAMD (SEQ ID NO:9). In yet a furtherembodiment, the polynucleotide encodes a polypeptide containing anHVR-H1 sequence comprising the sequence NISGTY (SEQ ID NO:7); an HVR-H2sequence containing the sequence RIYPSEGYTR (SEQ ID NO:8); and an HVR-H3sequence containing the sequence WVGVGFYAMD (SEQ ID NO:9).

In one embodiment, the host cell is prokaryotic and in anotherembodiment, the host cell is eukaryotic, such as a mammalian cell.

In a further aspect, the invention features a method of treating a tumorin a subject. This method includes administering to the subject anantibody or antibody fragment described herein, where the administeringis for a time and in an amount sufficient to treat or prevent the tumorin the subject. In one embodiment, the tumor is a colorectal tumor, abreast cancer, a lung cancer, a renal cell carcinoma, a glioma, aglioblastoma, or an ovarian cancer. In another embodiment, the antibodycontains an HVR-L1 sequence containing the sequence NIAKTISGY (SEQ IDNO:1) and specifically binds HER2 and VEGF. According to one embodiment,the antibody further contains one or two HVR sequences selected from (i)HVR-L2 containing the sequence WGSFLY (SEQ ID NO:2); and (ii) HVR-L3containing the sequence HYSSPP (SEQ ID NO:3). In another embodiment, theantibody contains, one, two, or three HVR sequences selected from (i)HVR-H1 containing the sequence NIKDTY (SEQ ID NO:4); (ii) HVR-H2containing the sequence RIYPTNGYTR (SEQ ID NO:5); and (iii) HVR-H3containing the sequence WGGDGFYAMD (SEQ ID NO:6). In an additionalembodiment, the antibody contains, one, two, or three HVR sequencesselected from (i) HVR-H1 containing the sequence NISGTY (SEQ ID NO:7);(ii) HVR-H2 containing the sequence RIYPSEGYTR (SEQ ID NO:8); and (iii)HVR-H3 containing the sequence WVGVGFYAMD (SEQ ID NO:9). In particularembodiments, the antibody comprises an HVR-L1 sequence containingNIAKTISGY (SEQ ID NO:1); an HVR-L2 sequence containing WGSFLY (SEQ IDNO:2); an HVR-L3 sequence containing HYSSPP (SEQ ID NO:3); an HVR-H1sequence containing NIKDTY (SEQ ID NO:4); an HVR-H2 sequence containingRIYPTNGYTR (SEQ ID NO:5); and an HVR-H3 sequence containing WGGDGFYAMD(SEQ ID NO:6) and specifically binds HER2 and VEGF. In anotherembodiment, the antibody contains an HVR-L1 sequence containingNIAKTISGY (SEQ ID NO:1); an HVR-L2 sequence containing WGSFLY (SEQ IDNO:2); an HVR-L3 sequence containing HYSSPP (SEQ ID NO:3); an HVR-H1sequence containing NISGTY (SEQ ID NO:7); an HVR-H2 sequence containingRIYPSEGYTR (SEQ ID NO:8); and an HVR-H3 sequence containing WVGVGFYAMD(SEQ ID NO:9) and specifically binds HER2 and VEGF.

In an embodiment, the method further includes administering to thesubject an additional anti-cancer therapy. In another embodiment, theadditional anti-cancer therapy includes another antibody, achemotherapeutic agent, a cytotoxic agent, an anti-angiogenic agent, animmunosuppressive agent, a prodrug, a cytokine, a cytokine antagonist,cytotoxic radiotherapy, a corticosteroid, an anti-emetic, a cancervaccine, an analgesic, or a growth-inhibitory agent.

In an additional embodiment, the additional anti-cancer therapy isadministered prior to or subsequent to the administration of anantibody. In a further embodiment, the additional anti-cancer therapy isadministered concurrently with an antibody.

In a further aspect, the invention features a method of treating anautoimmune disease in a subject. This method includes administering tothe subject an antibody or antibody fragment described herein, where theadministering is for a time and in an amount sufficient to treat orprevent the autoimmune disease in the subject. In one embodiment, theantibody contains an HVR-L1 sequence containing the sequence NIAKTISGY(SEQ ID NO:1) and specifically binds HER2 and VEGF. According to oneembodiment, the antibody contains one or two HVR sequences selected from(i) HVR-L2 containing the sequence WGSFLY (SEQ ID NO:2); and (ii) HVR-L3containing the sequence HYSSPP (SEQ ID NO:3). In another embodiment, theantibody contains, one, two, or three HVR sequences selected from (i)HVR-H1 containing the sequence NIKDTY (SEQ ID NO:4); (ii) HVR-H2containing the sequence RIYPTNGYTR (SEQ ID NO:5); and (iii) HVR-H3containing the sequence WGGDGFYAMD (SEQ ID NO:6). In an additionalembodiment, the antibody contains, one, two, or three HVR sequencesselected from (i) HVR-H1 containing the sequence NISGTY (SEQ ID NO:7);(ii) HVR-H2 containing the sequence RIYPSEGYTR (SEQ ID NO:8); and (iii)HVR-H3 containing the sequence WVGVGFYAMD (SEQ ID NO:9). In particularembodiments, the antibody contains an HVR-L1 sequence containingNIAKTISGY (SEQ ID NO:1); an HVR-L2 sequence containing WGSFLY (SEQ IDNO:2); an HVR-L3 sequence containing HYSSPP (SEQ ID NO:3); an HVR-H1sequence containing NIKDTY (SEQ ID NO:4); an HVR-H2 sequence containingRIYPTNGYTR (SEQ ID NO:5); and an HVR-H3 sequence containing WGGDGFYAMD(SEQ ID NO:6) and specifically binds HER2 and VEGF or the antibodycontains an HVR-L1 sequence comprising NIAKTISGY (SEQ ID NO:1); anHVR-L2 sequence containing WGSFLY (SEQ ID NO:2); an HVR-L3 sequencecontaining HYSSPP (SEQ ID NO:3); an HVR-H1 sequence containing NISGTY(SEQ ID NO:7); an HVR-H2 sequence containing RIYPSEGYTR (SEQ ID NO:8);and an HVR-H3 sequence containing WVGVGFYAMD (SEQ ID NO:9) andspecifically binds HER2 and VEGF.

In yet another aspect, the invention features a method of treating anon-malignant disease involving abnormal activation of HER2 in asubject. This method includes administering to the subject an antibodyor antibody fragment described herein, where the administering is for atime and in an amount sufficient to treat or prevent the non-malignantdisease in the subject. In one embodiment, the antibody contains anHVR-L1 sequence containing the sequence NIAKTISGY (SEQ ID NO:1) andspecifically binds HER2 and VEGF. According to one embodiment, theantibody comprises one or two HVR sequences selected from (i) HVR-L2containing the sequence WGSFLY (SEQ ID NO:2); and (ii) HVR-L3 containingthe sequence HYSSPP (SEQ ID NO:3). In another embodiment, the antibodyfurther contains, one, two, or three HVR sequences selected from (i)HVR-H1 containing the sequence NIKDTY (SEQ ID NO:4); (ii) HVR-H2containing the sequence RIYPTNGYTR (SEQ ID NO:5); and (iii) HVR-H3containing the sequence WGGDGFYAMD (SEQ ID NO:6). In an additionalembodiment, the antibody contains, one, two, or three HVR sequencesselected from (i) HVR-H1 containing the sequence NISGTY (SEQ ID NO:7);(ii) HVR-H2 containing the sequence RIYPSEGYTR (SEQ ID NO:8); and (iii)HVR-H3 containing the sequence WVGVGFYAMD (SEQ ID NO:9). In particularembodiments, the antibody contains an HVR-L1 sequence containingNIAKTISGY (SEQ ID NO:1); an HVR-L2 sequence containing WGSFLY (SEQ IDNO:2); an HVR-L3 sequence containing HYSSPP (SEQ ID NO:3); an HVR-H1sequence containing NIKDTY (SEQ ID NO:4); an HVR-H2 sequence containingRIYPTNGYTR (SEQ ID NO:5); and an HVR-H3 sequence containing WGGDGFYAMD(SEQ ID NO:6) and specifically binds HER2 and VEGF or the antibodycontains an HVR-L1 sequence comprising NIAKTISGY (SEQ ID NO:1); anHVR-L2 sequence containing WGSFLY (SEQ ID NO:2); an HVR-L3 sequencecontaining HYSSPP (SEQ ID NO:3); an HVR-H1 sequence containing NISGTY(SEQ ID NO:7); an HVR-H2 sequence containing RIYPSEGYTR (SEQ ID NO:8);and an HVR-H3 sequence containing WVGVGFYAMD (SEQ ID NO:9) andspecifically binds HER2 and VEGF.

Additional aspects of the invention feature the use of the antibodiesand antibody fragments described herein in the treatment of a tumor, anautoimmune disease, or a non-malignant disease involving abnormalactivation of HER2 in a subject, as well as use in the manufacture of amedicament for the treatment of a tumor, an autoimmune disease, or anon-malignant disease involving abnormal activation of HER2 in asubject. In one embodiment of these uses, the antibody contains anHVR-L1 sequence containing the sequence NIAKTISGY (SEQ ID NO:1) andspecifically binds HER2 and VEGF. According to one embodiment, theantibody further contains one or two HVR sequences selected from (i)HVR-L2 containing the sequence WGSFLY (SEQ ID NO:2); and (ii) HVR-L3containing the sequence HYSSPP (SEQ ID NO:3). In another embodiment, theantibody contains, one, two, or three HVR sequences selected from (i)HVR-H1 containing the sequence NIKDTY (SEQ ID NO:4); (ii) HVR-H2containing the sequence RIYPTNGYTR (SEQ ID NO:5); and (iii) HVR-H3containing the sequence WGGDGFYAMD (SEQ ID NO:6). In an additionalembodiment, the antibody contains, one, two, or three HVR sequencesselected from (i) HVR-H1 containing the sequence NISGTY (SEQ ID NO:7);(ii) HVR-H2 containing the sequence RIYPSEGYTR (SEQ ID NO:8); and (iii)HVR-H3 containing the sequence WVGVGFYAMD (SEQ ID NO:9). In particularembodiments, the antibody contains an HVR-L1 sequence containingNIAKTISGY (SEQ ID NO:1); an HVR-L2 sequence containing WGSFLY (SEQ IDNO:2); an HVR-L3 sequence containing HYSSPP (SEQ ID NO:3); an HVR-H1sequence containing NIKDTY (SEQ ID NO:4); an HVR-H2 sequence containingRIYPTNGYTR (SEQ ID NO:5); and an HVR-H3 sequence containing WGGDGFYAMD(SEQ ID NO:6) and specifically binds HER2 and VEGF or the antibodycontains an HVR-L1 sequence containing NIAKTISGY (SEQ ID NO:1); anHVR-L2 sequence containing WGSFLY (SEQ ID NO:2); an HVR-L3 sequencecontaining HYSSPP (SEQ ID NO:3); an HVR-H1 sequence containing NISGTY(SEQ ID NO:7); an HVR-H2 sequence containing RIYPSEGYTR (SEQ ID NO: 8);and an HVR-H3 sequence containing WVGVGFYAMD (SEQ ID NO:9) andspecifically binds HER2 and VEGF.

In an embodiment of the methods of treating a tumor, an autoimmunedisease, or a non-malignant disease involving abnormal activation ofHER2 described herein, the subject is a human.

Also, contemplated are kits, compositions, and articles of manufacturecomprising the antibodies and antibody fragments described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the designed diversity in various LC libraries.

FIG. 2 shows a summary of four light chain libraries used to alteranti-VEGF antibodies or anti-Her2 antibodies to bind to an additionaltarget. The italicized NNK and XYZ refer to codon sets. Ys, Ds, Ts andSs refer to soft randomizations by having tyrosine, aspartic acid,threonine and serine, respectively, occurring 50% of the time and anyone of the 20 amino acids occurring the other 50% of the time. D/Ds andT/Ts refer to a soft randomization having D or T, respectively,occurring 75% of the time and any one of the 20 amino acids occurringthe other 25% of the time.

FIG. 3 shows sequences of HC, LC CDR residues of light chain templates.Template CDR-L1, CDR-L2, FR3 (CDR-L4), and CDR-L3 sequences are listedas SEQ ID NOs: 86-106, as shown.

FIG. 4 shows the natural and designed diversity of light chain CDRs. Ateach position, the Herceptin® antibody sequence is shown in parenthesis.An “*” denotes an insertion not present in the Herceptin® antibody.

FIGS. 5A, 5B-1, and 5B-2 show the sequences of specific antigen-bindingclones isolated from the light chain (LC) library. FIG. 5A shows the LCCDR sequences (CDR-L1, CDR-L2, CDR-L3) of monospecific phage clonesbiding to VEGF, DR5, and Fc (SEQ ID NOs: 107-259), and FIG. 5B showsbispecific Fabs binding to VEGF/HER2, DR5/HER2, and Fc/HER2. CDR-L1,CDR-L2, and CDR-L3 sequences are shown as SEQ ID NOs: 260-331 and SEQ IDNOs: 332-412 in FIGS. 5B-1 and 5B-2, respectively. The light chainframework and heavy chain sequences correspond to that of the Herceptin®antibody with the exception of LC framework substitution R66G.

FIG. 6 is a graph showing binding specificity of the antibodies derivedfrom the LC library. The results for antibodies bH1, bH3, 3-1, bD1, bD2,4-1, and 4-5 are shown. Bound IgG antibodies were detectedspectrophotometrically (optical density at 450 nm, y-axis). The proteinsincluded in the assay were (left to right for each antibody) humanvascular endothelial growth factor A (hVEGF-A), hVEGF-C, hVEGF-D, hHER2extracellular domain (ECD), epidermal growth factor receptorextracellular domain (hEGFR), human death receptor 5 (hDR5), bovineserum albumin (BSA), casein, fetal bovine serum (FBS), WIL2 cell lysate,and NR6 cell lysate.

FIG. 7 shows sorting conditions and enrichment of Library C and D.

FIG. 8 shows VEGF binders, with CDR-L1 and CDR-L2 sequences shown as SEQID NOs: 413-474. Residues 28, 30, 30a, 31, 92, 93, and 93a were fullydiverse. Residues 32, 50, 53, 91 and 94 were restricted. Residues 29,33, and 51 were limited (<3).

FIG. 9 shows human VEGF binders, combined plate and solution selection.CDR-L1 (L1), CDR-L2 (L2), and CDR-L3 (L3) sequences are listed as SEQ IDNOs: 475-492, as shown.

FIGS. 10A and 10B show clones that bind both VEGF and HER2. CDR-L1 (L1),CDR-L2 (L2), and CDR-L3 (L3) sequences for FIGS. 10A and 10B are shownas SEQ ID NOs: 493-585 and SEQ ID NOs: 586-675, respectively.

FIG. 11 shows clones that only bind VEGF and lost the binding activitywith HER2. CDR-L1 (L1), CDR-L2 (L2), and CDR-L3 (L3) sequences are shownas SEQ ID NOs: 676-753.

FIG. 12 shows clones binding to VEGF.

FIGS. 13A and 13B show clones that block VEGF binding to VEGFR1-D2 orD1.

FIGS. 14A and 14B show VEGF binders and the affinities of VEGF bindersfrom library L1/L2/L3-C,D. CDR-L1 (L1), CDR-L2 (L2), and CDR-L3 (L3)sequences for FIG. 14A are listed as SEQ ID NOs: 754-780, as shown.

FIG. 15 shows clones that can bind both hVEGF and HER2. CDR-L1 (L1),CDR-L2 (L2), and CDR-L3 (L3) sequences are listed as SEQ ID NOs:781-794, as shown.

FIG. 16 shows the LC library binders used in scFv′2 formation anddisplayed on phage. CDR-L1 (L1), CDR-L2 (L2), and CDR-L3 (L3) sequencesare listed as SEQ ID NOs: 796-828, as shown.

FIG. 17 shows the expression of various clones in Fab or hIgG form.

FIGS. 18A and 18B show ELISAs of clones in hIgG form binding tohVEGF165.

FIG. 19 shows ELISAs of clones in hIgG form binding to immobilizedprotein targets.

FIG. 20 shows competitive ELISAs of clones in hIgG form in the presenceof Her2 and VEGF or DR5.

FIG. 21 shows a Biacore Analysis of binding to VEGF or HER2.

FIG. 22 shows binding to HER2-ECD or hVEGF with an IgG or Fab having alight chain obtained from a different binding clone.

FIGS. 23A and 23B show an anti-VEGF antibody blocking VEGF interactionwith VEGFR1 D 1-3 and KDR D1-7.

FIG. 24 shows antibodies blocking B20-4.1 and VEGF binding.

FIG. 25 shows antibodies blocking Avastin® antibody and VEGF binding.

FIG. 26 shows crystal structures of the bispecific bH1 Fab bound to HER2or VEGF.

FIG. 27 is a graph showing that anti-VEGF antibodies block hVEGF bindingto VEGF receptor 2 (VEGFR2).

FIG. 28 shows crystal structures of the bispecific bH1 Fab bound to HER2or VEGF.

FIG. 29 is a series of pie charts showing the individual CDRcontributions to the structural paratope for bH1. The paratope size forVEGF is 730 Å² and for HER2 is 690 Å². The heavy chain CDRs areindicated in gray and the light chain CDRs in white.

FIG. 30 shows the superposition of the CDR loops of VEGF/HER2-bound bH1or HER2-bound Herceptin® antibody in the same orientation as FIG. 28.

FIG. 31 shows crystal structures of the bispecific bH1 Fab bound to HER2or VEGF. CDR-L1 of the two bH1 complexes are shown in the sameorientation.

FIG. 32 shows the energetically important binding sites of bH1 for VEGFand HER2 binding.

FIG. 33 shows codons of bH1 that were shotgun scanned.

FIG. 34 shows a library consortium.

FIG. 35 shows an antibody clone with shotgun scan mutations screened bybinding to VEGF.

FIG. 36 shows an antibody clone with shotgun scan mutations screened bybinding to HER2.

FIGS. 37A-37D show alanine scanning results. FIGS. 37A and 37B show theresults of an alanine scan of bH1 for (FIG. 37A) VEGF binding or (FIG.37B) HER2 binding, and FIGS. 37C and 37D show the results of a homologscan of bH1 for (FIG. 37C) VEGF binding or (FIG. 37D) HER2 binding.

FIG. 38 shows alanine scanning results of bH1 or the Herceptin® antibodymutants.

FIGS. 39A-1, 39A-2, and 39A-3 show shotgun alanine and homolog scanningof bH1 Fab for binding to VEGF. FIGS. 39B-1, 39 B-2, and 39B-3 showshotgun alanine and homolog scanning of bH1 Fab for binding to HER2.

FIG. 40 shows the energetically important binding sites of bH1 for VEGFand HER2 binding.

FIG. 41 shows bH1 VEGF-affinity matured clone sequences and bindingaffinity for VEGF or HER2. CDR-L1, CDR-L2 (CDR 2), and CDR-L3 (CDR 3)sequences are listed as SEQ ID NOs: 829-885, as shown.

FIG. 42 shows the inhibition of VEGF induced HUVEC cell proliferationwith anti-VEGF antibodies.

FIG. 43 shows binding of bispecific antibodies to HER2 expressed on NR6cells.

FIG. 44 shows the results of competitive binding experiments for bH1 toVEGF or HER2.

FIG. 45 shows that bH1 and affinity improved variants bH1-44 and bH1-81IgG inhibit HER2 and VEGF-mediated cell proliferation in vitro.

FIG. 46 shows the binding specificity of bispecific antibodies derivedfrom the LC library.

FIGS. 47A and 47B show that anti-VEGF antibodies block VEGF binding toVEGFR2 receptors. FIG. 47A shows human VEGF binding and FIG. 47B showsmurineVEGF binding.

FIGS. 48A and 48B show that VEGF and HER2 compete for binding to bH1-44bispecific IgG in solution.

FIGS. 49A and 49B show that the bispecific antibodies bH1 and bH1-44bind to HER2 expressing mouse fibroblast cells (NR6; FIG. 49B), but notto HER2 negative NR6 cells (FIG. 49A).

FIG. 50 shows that the bispecific bH1 antibody specificallyimmunoprecipitates VEGF or HER2 from mouse fibroblast (NR6) lysates, butnot other proteins.

FIG. 51 shows tumor inhibition of bH1-44 in Colo205 and BT474M1xenografts in immuno-compromised mice.

FIGS. 52A, 52B, and 53 depict exemplary acceptor human consensusframework sequences for use in practicing the instant invention withsequence identifiers as follows:

Variable Heavy (VH) Consensus Frameworks (FIGS. 52A and 52B)

human VH subgroup I consensus framework regions FR1, FR2, FR3, and FR4minus Kabat CDRs (IA: SEQ ID NOS: 42-45, respectively)human VH subgroup I consensus framework regions FR1, FR2, FR3, and FR4minus extended hypervariable regions (IB: SEQ ID NOS: 46, 47, 44, and45, respectively; IC: SEQ ID NOS: 46-48 and 45, respectively; ID: SEQ IDNOS: 42, 47, 49, and 45, respectively) human VH subgroup II consensusframework regions FR1, FR2, FR3, and FR4 minus Kabat CDRs (IIA: SEQ IDNOS: 50-52 and 45, respectively)human VH subgroup II consensus framework regions FR1, FR2, FR3, and FR4minus extended hypervariable regions (IIB: SEQ ID NOS: 53, 54, 52, and45, respectively; IIC: SEQ ID NOS: 53-55 and 45, respectively; HD: SEQID NOS: 53, 54, 56, and 45, respectively)human VH subgroup III consensus framework regions FR1, FR2, FR3, and FR4minus Kabat CDRs (MA: SEQ ID NOS: 57-59 and 45, respectively)human VH subgroup III consensus framework regions FR1, FR2, FR3, and FR4minus extended hypervariable regions (MB: SEQ ID NOS: 60, 61, 59, and45, respectively; MC: SEQ ID NOS: 60-62 and 45, respectively; HID: SEQID NOS: 60, 61, 63, and 45, respectively)human VH acceptor framework regions FR1, FR2, FR3, and FR4 minus KabatCDRs (Acceptor A: SEQ ID NOS: 64, 58, 65, and 45, respectively)human VH acceptor framework regions FR1, FR2, FR3, and FR4 minusextended hypervariable regions (Acceptor B: SEQ ID NOS: 60, 61, 65, and45, respectively; Acceptor C: SEQ ID NOS: 60, 61, 66, and 45,respectively)human VH acceptor 2 framework regions FR1, FR2, FR3, and FR4 minus KabatCDRs (Second Acceptor A: SEQ ID NOS: 64, 58, 67, and 45, respectively)human VH acceptor 2 framework regions FR1, FR2, FR3, and FR4 minusextended hypervariable regions (Second Acceptor B: SEQ ID NOS: 60, 61,67, and 45, respectively;Second Acceptor C: SEQ ID NOS: 60, 61, 68, and 45, respectively; SecondAcceptor D: SEQ ID NOS: 60, 61, 69, and 45, respectively)

Variable Light (VL) Consensus Frameworks (FIG. 53)

human VL kappa subgroup I consensus framework regions FR1, FR2, FR3, andFR4 (kv1: SEQ ID NOS: 70-73, respectively)human VL kappa subgroup II consensus framework regions FR1, FR2, FR3,and FR4 (kv2: SEQ ID NOS: 74-76 and 73, respectively)human VL kappa subgroup III consensus framework regions FR1, FR2, andFR3 (kv3: SEQ ID NOS: 77-79 and 73, respectively)human VL kappa subgroup IV consensus framework regions FR1, FR2, and FR3(kv4: SEQ ID NOS: 80-82 and 73, respectively)

FIG. 54 shows the residues that make structural contacts or an energeticinteraction with HER2, VEGF, or both. The residues that make structuralcontacts (>25% buried) or an energetic interaction (ΔΔG>10% totalbinding energy) with HER2 (light grey), VEGF (grey), or both (shared,black) are mapped on the surface of HER2-bound bH1.

FIG. 55 shows the bH1/VEGF and bH1/HER2 binding interfaces. A close-upof the bH1/VEGF (A) and the bH1/HER2 (B) binding interface illustratesthe structural differences between VEGF and HER2 in the regions ofantibody binding. Surface representations of VEGF (C) and HER2-ECD (D)are shown in the same orientation relative to bH1 Fab. The residues incontact with bH1 Fab (closer than 4.5 Å) are highlighted. There is noapparent similarity between the two epitopes for bH1 in terms ofchemical composition or topology.

FIG. 56 shows that bH1 and bH1-44 antibodies block human VEGF binding toVEGFR1. Biotinylated human VEGF₁₆₅ was incubated with increasingconcentrations of IgG (x-axis), then captured on immobilized humanVEGFR1-Fc, and detected with horseradish peroxidase-conjugatedstreptavidin with added substrate (normalized % OD₄₅₀, y-axis).

FIG. 57 shows alanine scanning results of bH1 and bH1-44 mutants.Alanine scanning mutagenesis identified the functionally importantresidues for VEGF and/or HER2 binding. F values represent the relativecontribution of each scanned residue to antigen binding. F values weredetermined for bH1-44 binding to VEGF and HER2 (black bars), andcompared to the F values of bH1 (white bars). The amino acids inparenthesis denote bH1-44 residues that differ from bH1. This graph wasadapted from FIG. 56.

FIG. 58 shows the binding of bH1-44 I29A Y32A bH1-44 and R50A R58AbH1-44 antibodies to VEGF and HER2. The ELISA binding assays show theability of bH1-44 IgG and the two double mutants to bind to biotinylatedVEGF₁₀₉ (left) or HER2-ECD (right), and compete with the immobilizedanti-VEGF antibody or Herceptin, respectively. The I29A/Y32A LC mutanthas lost binding of VEGF, while maintaining similar affinity for HER2 asbH1-44. The R50A/R58A HC mutant has lost affinity for HER2, but retainsVEGF binding.

FIGS. 59A-59E show the calorimetric measurements of the enthalpy changesassociated with antigen binding. FIGS. 59A-59F show the data for bH1binding to VEGF (FIG. 59A), bH1 binding to HER2 (FIG. 59B), bH1-44binding to VEGF (FIG. 59C), bH1-44 binding to HER2 (FIG. 59D), bH1-44HC-R50A+R58A binding to VEGF (FIG. 59E), and bH1-44 LC-I29A+Y32A bindingto HER2 (FIG. 59F). The figures show the individual heat pulses (top)and the heats of reaction (bottom), which are calculated by integrationof each pulse, plotted as a function of the antibody to antigen ratio atthe end of the injections. The small magnitude of the enthalpy changesrequired relatively high protein concentrations, which precludedaccurate estimation of K_(D) when the affinity was high. FIGS. 59A-59D:Solutions of VEGF₁₀₉ of HER2-ECD at concentrations ranging from 10-20 μMwere titrated by 15 injections of bH1 or bH1-44 Fab at concentrationsfrom 100 to 200 μM. FIGS. 59E-59F: Solutions of VEGF₁₀₉ or HER2-ECD atconcentrations of 10 to 20 μM were titrated by 20 injections of bH1-44LC-I29A+Y32A Fab or bH1-44 HC-R50A+R58A Fab at concentrations of 150 and250 Titrations number 1 and 13 in (FIG. 59E) were excluded from theanalysis due to instrument noise.

FIG. 60 shows the thermodynamic profiles of the bH1 variants and theHerceptin® antibody. Each dual specific variant (bH1, bH1-81, andbH1-44) has thermodynamic profiles characterized by favorable enthalpyand entropy for both VEGF and HER2 binding. The variants HC-R50A+R58Aand LC-129A+Y32A that have lost affinity for HER2 or VEGF respectively,display similar thermodynamic profiles as bH1-44. The thermodynamicprofiles of the bH1-44/HER2 interaction are distinct fromHerceptin/HER2.

FIG. 61 shows the comparison of the bH1, bH1-44, and the Herceptin®antibody hotspots for HER2 binding based on the alanine scanningmutagenesis data. Hotspot residues are highlighted in grey mapped ontothe Herceptin® (Herceptin) structure or bH1 Fab structures (bH1,bH1-44). Hotspots are defined as ΔΔG greater than or equal to 10% of thetotal binding free energy (ΔG). The structural contact sites (within 4.5Å of the antigens in the structures) are outlined by light dotted lines.The HC and LC are separated by a black dotted line. The underlinedresidues differ in sequence from Herceptin®.

FIG. 62 shows the estimated heat capacity changes associated with bH1-44Fab binding with VEGF or HER2. ΔCp was determined from the slope of thetemperature dependence of AH between 20 and 37° C. Over this range, ΔCpappears to be independent of T, based on the linear relationship betweenΔH and T (R=0.991 for bH1-44/HER2, R=0.9989 for bH1-44/VEGF). The ΔCpfor Herceptin®/HER2 was previously determined by Kelley et al.(Biochemistry, 1992).

FIGS. 63A and 63B show the binding kinetics of bH1-44 variants measuredby BIAcore. The figures show overlays of representative response versustime plots for the binding interactions between immobilized (A) VEGF₁₀₉or (B) HER2-ECD and 0.5 μM solutions of bH1-44 Fab (red), bH1-44-LC-Y32(green), bH1-44-LC-I29A+Y32A (magenta), and bH1-44-HC-R50A+R58A (grey).The traces represent binding to the same immobilized CM5 chip, which wasregenerated after each Fab run. No binding was detected forbH1-44-LC-I29A+Y32A to VEGF or for bH1-44-HC-R50A+R58A to HER2 at 0.5μM. The variant bH1-44-Y32A displayed significantly weakened binding toVEGF compared to the wild type bH1-44.

FIGS. 64A-64D show the mapping of the specificity determining residuesof bH1-44 on the crystal structure of bH1. The residues that areimportant for VEGF binding (LC-I29 and LC-Y32: FIGS. 64A and 64B) andthe residues that are important for HER binding (HC-R50 and HC-R58;FIGS. 64C and 64D) are shown in dark grey as sticks on the bH1/VEGF(FIGS. 64A and 64C, 2.6 Å resolution) or bH1/HER2 (FIGS. 64B and 64D,2.9 Å resolution) crystal structures. The residues 129 and Y32 appear tobe involved in intra-chain interactions that serve to maintain theCDR-L1 loop conformation necessary for VEGF-binding. 129 is solventexposed in the HER2 structure. Y32 packs against HER2, but does notengage in productive antigen contact. R50 and R58 pack against D560 andE558 on HER2, and appear to engage in charge-charge interactions. R50and R58 are solvent exposed in the VEGF solvent structure.

FIG. 65 shows the expression of the Herceptin® mutant Fabs (R50A, R58A,and R50A/R58A).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods of making multispecificantibodies and antibody fragments, as well as antibodies identifiedusing these methods and their use. In general, the methods of theinvention involve diversifying the light chain variable domain or theheavy chain variable domain of an antibody to generate variants that canbe stably expressed in a library. Diversified antibodies that arecapable of specifically binding two epitopes are then selected from thislibrary and further characterized.

Exemplary antibodies identified using the methods of the inventioninclude antibodies that bind both HER2 (human epidermal growth factorreceptor 2) and VEGF (vascular endothelial growth factor). Inparticular, the data described herein, for instance, in the belowExamples, show that mutations in the light chain complementaritydetermining regions (CDRs) of a HER2 antibody confer dual bindingcapabilities for unrelated protein antigens as well as HER2. Onebi-specific high affinity HER2/VEGF antibody is extensivelycharacterized. In addition, the crystal structures of this bi-specificFab in complex with HER2 and VEGF are shown and the energeticcontribution of the Fab residues by mutagenesis is evaluated. Thebinding sites for the two antigens overlap extensively; most of the CDRresidues that contact HER2 also engage VEGF. Energetically, however, theresidues of the heavy chain dominate the HER2 specificity while thelight chain dominates VEGF specificity.

The HER2/VEGF bi-specific antibody inhibits both HER2 and VEGF-mediatedcell proliferation in vitro and in vivo. These results demonstrate thataltering the sequence of the light chain variable domain of an antibodycan generate antibodies with dual specificity and function. For example,bH1-44 and bH1-81 have the potential to target two mechanisms of tumorprogression: tumor cell proliferation mediated by HER2 and tumorangiogenesis mediated by VEGF. Co-targeting two antigens with a singleantibody is an alternative to combination therapy.

I. Definitions

The term “multispecific antibody” is used in the broadest sense andspecifically covers an antibody comprising a heavy chain variable domain(V_(H)) and a light chain variable domain (V_(L)), where the V_(H)V_(L)unit has polyepitopic specificity (i.e., is capable of binding to twodifferent epitopes on one biological molecule or each epitope on adifferent biological molecule). Such multispecific antibodies include,but are not limited to, full length antibodies, antibodies having two ormore V_(L) and V_(H) domains, antibody fragments such as Fab, Fv, dsFv,scFv, diabodies, bispecific diabodies and triabodies, antibody fragmentsthat have been linked covalently or non-covalently. “Polyepitopicspecificity” refers to the ability to specifically bind to two or moredifferent epitopes on the same or different target(s). “Monospecific”refers to the ability to bind only one epitope. According to oneembodiment the multispecific antibody is an IgG1 form binds to eachepitope with an affinity of 5 μM to 0.001 pM, 3 μM to 0.001 pM, 1 μM to0.001 pM, 0.5 μM to 0.001 pM or 0.1 μM to 0.001 pM.

The basic 4-chain antibody unit is a heterotetrameric glycoproteincomposed of two identical light (L) chains and two identical heavy (H)chains (an IgM antibody consists of 5 of the basic heterotetramer unitsalong with an additional polypeptide called J chain, and thereforecontains 10 antigen binding sites, while secreted IgA antibodies canpolymerize to form polyvalent assemblages comprising 2-5 of the basic4-chain units along with J chain). In the case of IgGs, the 4-chain unitis generally about 150,000 daltons. Each L chain is linked to an H chainby one covalent disulfide bond, while the two H chains are linked toeach other by one or more disulfide bonds depending on the H chainisotype. Each H and L chain also has regularly spaced intrachaindisulfide bridges. Each H chain has, at the N-terminus, a variabledomain (V_(H)) followed by three constant domains (C_(H)) for each ofthe α and γ chains and four C_(H) domains for μ and ε isotypes. Each Lchain has, at the N-terminus, a variable domain (V_(L)) followed by aconstant domain (C_(L)) at its other end. The V_(L) is aligned with theV_(H) and the C_(L) is aligned with the first constant domain of theheavy chain (C_(H)1). Particular amino acid residues are believed toform an interface between the light chain and heavy chain variabledomains. The pairing of a V_(H) and V_(L) together forms a singleantigen-binding site. For the structure and properties of the differentclasses of antibodies, see, e.g., Basic and Clinical Immunology, 8thedition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.),Appleton & Lange, Norwalk, Conn., 1994, page 71 and Chapter 6.

The L chain from any vertebrate species can be assigned to one of twoclearly distinct types, called kappa and lambda, based on the amino acidsequences of their constant domains. Depending on the amino acidsequence of the constant domain of their heavy chains (C_(H)),immunoglobulins can be assigned to different classes or isotypes. Thereare five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, havingheavy chains designated α, δ, γ, ε, and μ, respectively. The γ and αclasses are further divided into subclasses on the basis of relativelyminor differences in C_(H) sequence and function, e.g., humans expressthe following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

The term “variable” refers to the fact that certain segments of thevariable domains differ extensively in sequence among antibodies. The Vdomain mediates antigen binding and defines specificity of a particularantibody for its particular antigen. However, the variability is notevenly distributed across the 110-amino acid span of the variabledomains. Instead, the V regions consist of relatively invariantstretches called framework regions (FRs) of 15-30 amino acids separatedby shorter regions of extreme variability called “hypervariable regions”that are each 9-12 amino acids long. The variable domains of nativeheavy and light chains each comprise four FRs, largely adopting abeta-sheet configuration, connected by three hypervariable regions,which form loops connecting, and in some cases forming part of, thebeta-sheet structure. The hypervariable regions in each chain are heldtogether in close proximity by the FRs and, with the hypervariableregions from the other chain, contribute to the formation of theantigen-binding site of antibodies (see Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). The constantdomains are not involved directly in binding an antibody to an antigen,but exhibit various effector functions, such as participation of theantibody in antibody dependent cellular cytotoxicity (ADCC).

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody which are responsible for antigen-binding.The hypervariable region generally comprises amino acid residues from a“complementarity determining region” or “CDR” (e.g., around aboutresidues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the V_(L), and aroundabout residues 26-35 (H1), 50-65 (H2) and 95-102 (H3) in the V_(H) (inone embodiment, H1 is around about residues 31-35); Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991)) and/orthose residues from a “hypervariable loop” (e.g., residues 26-32 (L1),50-52 (L2), and 91-96 (L3) in the V_(L), and 26-32 (H1), 53-55 (H2), and96-101 (H3) in the V_(H); Chothia and Lesk, J. Mol. Biol. 196:901-917(1987)).

“Framework regions” (FR) are those variable domain residues other thanthe CDR residues. Each variable domain typically has four FRs identifiedas FR1, FR2, FR3 and FR4. If the CDRs are defined according to Kabat,the light chain FR residues are positioned at about residues 1-23(LCFR1), 35-49 (LCFR2), 57-88 (LCFR3), and 98-107 (LCFR4) and the heavychain FR residues are positioned about at residues 1-30 (HCFR1), 36-49(HCFR2), 66-94 (HCFR3), and 103-113 (HCFR4) in the heavy chain residues.If the CDRs comprise amino acid residues from hypervariable loops, thelight chain FR residues are positioned about at residues 1-25 (LCFR1),33-49 (LCFR2), 53-90 (LCFR3), and 97-107 (LCFR4) in the light chain andthe heavy chain FR residues are positioned about at residues 1-25(HCFRI), 33-52 (HCFR2), 56-95 (HCFR3), and 102-113 (HCFR4) in the heavychain residues. In some instances, when the CDR comprises amino acidsfrom both a CDR as defined by Kabat and those of a hypervariable loop,the FR residues will be adjusted accordingly. For example, when CDRH1includes amino acids H26-H35, the heavy chain FR1 residues are atpositions 1-25 and the FR2 residues are at positions 36-49.

A “human consensus framework” is a framework which represents the mostcommonly occurring amino acid residues in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat. In one embodiment, for the VL, the subgroup is subgroup kappaI as in Kabat. In one embodiment, for the VH, the subgroup is subgroupIII as in Kabat.

The term “monoclonal antibody” as used herein refers to an antibody froma population of substantially homogeneous antibodies, i.e., theindividual antibodies comprising the population are substantiallysimilar and bind the same epitope(s), except for possible variants thatmay arise during production of the monoclonal antibody, such variantsgenerally being present in minor amounts. Such monoclonal antibodytypically includes an antibody comprising a variable region that binds atarget, wherein the antibody was obtained by a process that includes theselection of the antibody from a plurality of antibodies. For example,the selection process can be the selection of a unique clone from aplurality of clones, such as a pool of hybridoma clones, phage clones orrecombinant DNA clones. It should be understood that the selectedantibody can be further altered, for example, to improve affinity forthe target, to humanize the antibody, to improve its production in cellculture, to reduce its immunogenicity in vivo, to create a multispecificantibody, etc., and that an antibody comprising the altered variableregion sequence is also a monoclonal antibody of this invention. Inaddition to their specificity, the monoclonal antibody preparations areadvantageous in that they are typically uncontaminated by otherimmunoglobulins. The modifier “monoclonal” indicates the character ofthe antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method. For example, themonoclonal antibodies to be used in accordance with the presentinvention may be made by a variety of techniques, including thehybridoma method (e.g., Kohler et al., Nature, 256:495 (1975); Harlow etal., Antibodies: A Laboratory Manual, (Cold Spring Harbor LaboratoryPress, 2nd ed. 1988); Hammerling et al., in: Monoclonal Antibodies andT-Cell Hybridomas 563-681, (Elsevier, N.Y., 1981), recombinant DNAmethods (see, e.g., U.S. Pat. No. 4,816,567), phage display technologies(see, e.g., Clackson et al., Nature, 352:624-628 (1991); Marks et al.,J. Mol. Biol., 222:581-597 (1991); Sidhu et al., J. Mol. Biol.338(2):299-310 (2004); Lee et al., J. Mol. Biol. 340(5):1073-1093(2004); Fellouse, Proc. Nat. Acad. Sci. USA 101(34):12467-12472 (2004);and Lee et al. J. Immunol. Methods 284(1-2):119-132 (2004) andtechnologies for producing human or human-like antibodies from animalsthat have parts or all of the human immunoglobulin loci or genesencoding human immunoglobulin sequences (see, e.g., WO98/24893,WO/9634096, WO/9633735, and WO/91 10741, Jakobovits et al., Proc. Natl.Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258(1993); Bruggemann et al., Year in Immuno., 7:33 (1993); U.S. Pat. Nos.5,545,806, 5,569,825, 5,591,669 (all of GenPharm); U.S. Pat. No.5,545,807; WO 97/17852, U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825;5,625,126; 5,633,425; and 5,661,016, and Marks et al., Bio/Technology,10: 779-783 (1992); Lonberg et al., Nature, 368:856-859 (1994);Morrison, Nature, 368:812-813 (1994); Fishwild et al., NatureBiotechnology, 14:845-851 (1996); Neuberger, Nature Biotechnology, 14:826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol., 13:65-93(1995).

An “intact” antibody is one which comprises an antigen-binding site aswell as a C_(L) and at least heavy chain constant domains, C_(H)1,C_(H)2, and C_(H)3. The constant domains can be native sequence constantdomains (e.g., human native sequence constant domains) or amino acidsequence variant thereof. Preferably, the intact antibody has one ormore effector functions.

“Antibody fragments” comprise a portion of an intact antibody,preferably the antigen binding or variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)₂, andFv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870,Example 2; Zapata et al., Protein Eng. 8(10): 1057-1062 (1995));single-chain antibody molecules; and multispecific antibodies formedfrom antibody fragments. The expression “linear antibodies” generallyrefers to the antibodies described in Zapata et al., Protein Eng.,8(10):1057-1062 (1995). Briefly, these antibodies comprise a pair oftandem Fd segments (V_(H)-C_(H)1-V_(H)-C_(H)1) which, together withcomplementary light chain polypeptides, form a pair of antigen bindingregions. Linear antibodies can be bispecific or monospecific.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, and a residual “Fc” fragment, adesignation reflecting the ability to crystallize readily. The Fabfragment consists of an entire L chain along with the variable regiondomain of the H chain (V_(H)), and the first constant domain of oneheavy chain (C_(H)1). Pepsin treatment of an antibody yields a singlelarge F(ab′)₂ fragment which roughly corresponds to two disulfide linkedFab fragments having divalent antigen-binding activity and is stillcapable of cross-linking antigen. Fab′ fragments differ from Fabfragments by having additional few residues at the carboxy terminus ofthe C_(H)1 domain including one or more cysteines from the antibodyhinge region. Fab′-SH is the designation herein for Fab′ in which thecysteine residue(s) of the constant domains bear a free thiol group.F(ab′)₂ antibody fragments originally were produced as pairs of Fab′fragments which have hinge cysteines between them. Other chemicalcouplings of antibody fragments are also known.

The Fc fragment comprises the carboxy-terminal portions of both H chainsheld together by disulfides. The effector functions of antibodies aredetermined by sequences in the Fc region; this region is also the partrecognized by Fc receptors (FcR) found on certain types of cells.

“Fv” consists of a dimer of one heavy- and one light-chain variableregion domain in tight, non-covalent association. From the folding ofthese two domains emanate six hypervariable loops (3 loops each from theH and L chain) that contribute the amino acid residues for antigenbinding and confer antigen binding specificity to the antibody. However,even a single variable domain (or half of an Fv comprising only threeCDRs specific for an antigen) has the ability to recognize and bindantigen, although often at a lower affinity than the entire bindingsite.

“Single-chain Fv” also abbreviated as “sFv” or “scFv” are antibodyfragments that comprise the V_(H) and V_(L) antibody domains connectedinto a single polypeptide chain. Preferably, the sFv polypeptide furthercomprises a polypeptide linker between the V_(H) and V_(L) domains whichenables the sFv to form the desired structure for antigen binding. For areview of sFv, see Pluckthun in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315 (1994); Borrebaeck 1995.

The term “diabodies” refers to small antibody fragments prepared byconstructing sFv fragments (see preceding paragraph) with short linkers(about 5-10 residues) between the V_(H) and V_(L) domains such thatinter-chain but not intra-chain pairing of the V domains is achieved,resulting in a bivalent fragment, i.e., fragment having twoantigen-binding sites. Bispecific diabodies are heterodimers of two“crossover” sFv fragments in which the V_(H) and V_(L) domains of thetwo antibodies are present on different polypeptide chains. Diabodiesare described more fully in, for example, EP 404,097; WO 93/11161; andHollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

“Humanized” forms of non-human (e.g., rodent) antibodies are chimericantibodies that contain minimal sequence derived from the non-humanantibody. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or non-human primate having the desired antibodyspecificity, affinity, and capability. In some instances, frameworkregion (FR) residues of the human immunoglobulin are replaced bycorresponding non-human residues. Furthermore, humanized antibodies cancomprise residues that are not found in the recipient antibody or in thedonor antibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992).

As used herein, “codon set” refers to a set of different nucleotidetriplet sequences used to encode desired variant amino acids. A set ofoligonucleotides can be synthesized, for example, by solid phasesynthesis, including sequences that represent all possible combinationsof nucleotide triplets provided by the codon set and that will encodethe desired group of amino acids. A standard form of codon designationis that of the IUB code, which is known in the art and described herein.A codon set typically is represented by 3 capital letters in italics,e.g., NNK, NNS, XYZ, DVK, and the like (e.g., NNK codon refers toN=A/T/G/C at positions 1 and 2 in the codon and K=G/T at equimolar ratioin position 3 to encode all 20 natural amino acids). A “non-random codonset”, as used herein, thus refers to a codon set that encodes selectamino acids that fulfill partially, preferably completely, the criteriafor amino acid selection as described herein. Synthesis ofoligonucleotides with selected nucleotide “degeneracy” at certainpositions is well known in that art, for example the TRIM approach(Knappek et al., J. Mol. Biol. 296:57-86, 1999); Garrard and Henner,Gene 128:103, 1993). Such sets of oligonucleotides having certain codonsets can be synthesized using commercial nucleic acid synthesizers(available from, for example, Applied Biosystems, Foster City, Calif.),or can be obtained commercially (for example, from Life Technologies,Rockville, Md.). Therefore, a set of oligonucleotides synthesized havinga particular codon set will typically include a plurality ofoligonucleotides with different sequences, the differences establishedby the codon set within the overall sequence. Oligonucleotides, as usedaccording to the invention, have sequences that allow for hybridizationto a variable domain nucleic acid template and also can, but do notnecessarily, include restriction enzyme sites useful for, for example,cloning purposes.

An antibody of this invention “which binds” an antigen of interest isone that binds the antigen with sufficient affinity such that theantibody is useful as a diagnostic and/or therapeutic agent in targetinga protein or a cell or tissue expressing the antigen, and does notsignificantly cross-react with other proteins. In such embodiments, theextent of binding of the antibody to a “non-target” protein will be lessthan about 10% of the binding of the antibody to its particular targetprotein as determined by fluorescence activated cell sorting (FACS)analysis or radioimmunoprecipitation (RIA) or ELISA. With regard to thebinding of an antibody to a target molecule, the term “specific binding”or “specifically binds to” or is “specific for” a particular polypeptideor an epitope on a particular polypeptide target means binding that ismeasurably different from a non-specific interaction (e.g., for bH1-44or bH1-81, a non-specific interaction is binding to bovine serumalbumin, casein, fetal bovine serum, or neuravidin). Specific bindingcan be measured, for example, by determining binding of a moleculecompared to binding of a control molecule. For example, specific bindingcan be determined by competition with a control molecule that is similarto the target, for example, an excess of non-labeled target. In thiscase, specific binding is indicated if the binding of the labeled targetto a probe is competitively inhibited by excess unlabeled target. Theterm “specific binding” or “specifically binds to” or is “specific for”a particular polypeptide or an epitope on a particular polypeptidetarget as used herein can be exhibited, for example, by a moleculehaving a Kd for the target of at least about 200 nM, alternatively atleast about 150 nM, alternatively at least about 100 nM, alternativelyat least about 60 nM, alternatively at least about 50 nM, alternativelyat least about 40 nM, alternatively at least about 30 nM, alternativelyat least about 20 nM, alternatively at least about 10 nM, alternativelyat least about 8 nM, alternatively at least about 6 nM, alternatively atleast about 4 nM, alternatively at least about 2 nM, alternatively atleast about 1 nM, or greater. In one embodiment, the term “specificbinding” refers to binding where a molecule binds to a particularpolypeptide or epitope on a particular polypeptide without substantiallybinding to any other polypeptide or polypeptide epitope.

“Binding affinity” generally refers to the strength of the sum total ofnoncovalent interactions between a single binding site of a molecule(e.g., an antibody) and its binding partner (e.g., an antigen). Unlessindicated otherwise, as used herein, “binding affinity” refers tointrinsic binding affinity which reflects a 1:1 interaction betweenmembers of a binding pair (e.g., antibody and antigen). The affinity ofa molecule X for its partner Y can generally be represented by thedissociation constant (Kd). Desirably the Kd is about 200 nM, 150 nM,100 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 8 nM, 6 nM, 4 nM, 2nM, 1 nM, or stronger. Affinity can be measured by common methods knownin the art, including those described herein. Low-affinity antibodiesgenerally bind antigen slowly and tend to dissociate readily, whereashigh-affinity antibodies generally bind antigen faster and tend toremain bound longer. A variety of methods of measuring binding affinityare known in the art, any of which can be used for purposes of thepresent invention.

In one embodiment, the “Kd” or “Kd value” according to this invention ismeasured by using surface plasmon resonance assays using a BIAcore™-2000or a BIAcore™-3000 (BIAcore, Inc., Piscataway, N.J.) at 25° C. withimmobilized antigen CM5 chips at ˜10 response units (RU). Briefly,carboxymethylated dextran biosensor chips (CM5, BIAcore Inc.) areactivated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to thesupplier's instructions. Antigen is diluted with 10 mM sodium acetate,pH 4.8, into 5 μg/ml (˜0.2 μM) before injection at a flow rate of 5μl/minute to achieve approximately 10 response units (RU) of coupledprotein. Following the injection of antigen, 1M ethanolamine is injectedto block unreacted groups. For kinetics measurements, two-fold serialdilutions of Fab (e.g., 0.78 nM to 500 nM) are injected in PBS with0.05% Tween 20 (PBST) at 25° C. at a flow rate of approximately 25μl/min Association rates (k_(on)) and dissociation rates (k_(off)) arecalculated using a simple one-to-one Langmuir binding model (BIAcoreEvaluation Software version 3.2) by simultaneous fitting the associationand dissociation sensorgram. The equilibrium dissociation constant (Kd)is calculated as the ratio k_(off)/k_(on). See, e.g., Chen, Y., et al.,(1999) J. Mol. Biol. 293:865-881. If the on-rate exceeds 10⁶ M⁻¹ S⁻¹ bythe surface plasmon resonance assay above, then the on-rate can bedetermined by using a fluorescent quenching technique that measures theincrease or decrease in fluorescence emission intensity (excitation=295nm; emission=340 nm, 16 nm band-pass) at 25° C. of a 20 nM anti-antigenantibody (Fab form) in PBS, pH 7.2, in the presence of increasingconcentrations of antigen as measured in a spectrometer, such as astop-flow equipped spectrophometer (Aviv Instruments) or a 8000-seriesSLM-Aminco spectrophotometer (ThermoSpectronic) with a stir red cuvette.

An “on-rate” or “rate of association” or “association rate” or “k_(on)”according to this invention can also be determined with the same surfaceplasmon resonance technique described above using a BIAcore™-2000 or aBIAcore™-3000 (BIAcore, Inc., Piscataway, N.J.) at 25° C. withimmobilized antigen CM5 chips at ˜10 response units (RU). Briefly,carboxymethylated dextran biosensor chips (CM5, BIAcore Inc.) areactivated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to thesupplier's instructions. Antigen is diluted with 10 mM sodium acetate,pH 4.8, into 5 μg/ml (˜0.2 μM) before injection at a flow rate of5μ/minute to achieve approximately 10 response units (RU) of coupledprotein. Following the injection of antigen, 1M ethanolamine is injectedto block unreacted groups. For kinetics measurements, two-fold serialdilutions of Fab (e.g., 0.78 nM to 500 nM) are injected in PBS with0.05% Tween 20 (PBST) at 25° C. at a flow rate of approximately 25μl/min Association rates (k_(on)) and dissociation rates (k_(off)) arecalculated using a simple one-to-one Langmuir binding model (BIAcoreEvaluation Software version 3.2) by simultaneous fitting the associationand dissociation sensorgram. The equilibrium dissociation constant (Kd)is calculated as the ratio k_(off)/k_(on). See, e.g., Chen, Y., et al.,(1999) J. Mol. Biol. 293:865-881. However, if the on-rate exceeds 10⁶M⁻¹ S⁻¹ by the surface plasmon resonance assay above, then the on-rateis preferably determined by using a fluorescent quenching technique thatmeasures the increase or decrease in fluorescence emission intensity(excitation=295 nm; emission=340 nm, 16 nm band-pass) at 25° C. of a 20nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence ofincreasing concentrations of antigen as measured in a spectrometer, suchas a stop-flow equipped spectrophometer (Aviv Instruments) or a8000-series SLM-Aminco spectrophotometer (ThermoSpectronic) with astirred cuvette.

“Biologically active” and “biological activity” and “biologicalcharacteristics” with respect to a polypeptide of this invention meanshaving the ability to bind to a biological molecule, except wherespecified otherwise.

“Biological molecule” refers to a nucleic acid, a protein, acarbohydrate, a lipid, and combinations thereof. In one embodiment, thebiologic molecule exists in nature.

“Isolated,” when used to describe the various antibodies disclosedherein, means an antibody that has been identified and separated and/orrecovered from a cell or cell culture from which it was expressed.Contaminant components of its natural environment are materials thatwould typically interfere with diagnostic or therapeutic uses for thepolypeptide, and can include enzymes, hormones, and other proteinaceousor non-proteinaceous solutes. In preferred embodiments, the antibodywill be purified (1) to a degree sufficient to obtain at least 15residues of N-terminal or internal amino acid sequence by use of aspinning cup sequenator, or (2) to homogeneity by SDS-PAGE undernon-reducing or reducing conditions using Coomassie blue or, preferably,silver stain. Isolated antibody includes antibodies in situ withinrecombinant cells, because at least one component of the polypeptidenatural environment will not be present. Ordinarily, however, isolatedpolypeptide will be prepared by at least one purification step.

The term “control sequences” refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

“Percent (%) amino acid sequence identity” with respect to thepolypeptide sequences identified herein is defined as the percentage ofamino acid residues in a candidate sequence that are identical with theamino acid residues in the polypeptide being compared, after aligningthe sequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity, and not considering any conservativesubstitutions as part of the sequence identity. Alignment for purposesof determining percent amino acid sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software such as BLAST, BLAST-2, ALIGN orMegalign (DNASTAR) software. Those skilled in the art can determineappropriate parameters for measuring alignment, including any algorithmsneeded to achieve maximal alignment over the full length of thesequences being compared. For purposes herein, however, % amino acidsequence identity values are generated using the sequence comparisoncomputer program ALIGN-2. The ALIGN-2 sequence comparison computerprogram was authored by Genentech, Inc. and the source code has beenfiled with user documentation in the U.S. Copyright Office, WashingtonD.C., 20559, where it is registered under U.S. Copyright RegistrationNo. TXU510087. The ALIGN-2 program is publicly available throughGenentech, Inc., South San Francisco, Calif. The ALIGN-2 program shouldbe compiled for use on a UNIX operating system, preferably digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

The amino acid sequences described herein are contiguous amino acidsequences unless otherwise specified.

“Structurally unsimilar” biological molecules according to thisinvention refers to biological molecules that are not in the same class(protein, nucleic acid, lipid, carbohydrates, etc.) or, for example,when referring to proteins, having less than 60% amino acid identity,less than 50% amino acid identity, less than 40% amino acid identity,less than 30% amino acid identity, less than 20% amino acid identity orless than 10% amino acid identity compared to each other.

“Stringency” of hybridization reactions is readily determinable by oneof ordinary skill in the art, and generally is an empirical calculationdependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured DNA toreanneal when complementary strands are present in an environment belowtheir melting temperature. The higher the degree of desired homologybetween the probe and hybridizable sequence, the higher the relativetemperature which can be used. As a result, it follows that higherrelative temperatures would tend to make the reaction conditions morestringent, while lower temperatures less so. For additional details andexplanation of stringency of hybridization reactions, see Ausubel etal., Current Protocols in Molecular Biology, Wiley IntersciencePublishers, (1995).

“Stringent conditions” or “high stringency conditions”, as definedherein, can be identified by those that: (1) employ low ionic strengthand high temperature for washing, for example 0.015 M sodiumchloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.;(2) employ during hybridization a denaturing agent, such as formamide,for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1%Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (3)overnight hybridization in a solution that employs 50% formamide, 5×SSC(0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8),0.1% sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon spermDNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with a 10minute wash at 42° C. in 0.2×SSC (sodium chloride/sodium citrate)followed by a 10 minute high-stringency wash consisting of 0.1×SSCcontaining EDTA at 55° C.

“Moderately stringent conditions” can be identified as described bySambrook et al., Molecular Cloning: A Laboratory Manual, New York: ColdSpring Harbor Press, 1989, and include the use of washing solution andhybridization conditions (e.g., temperature, ionic strength, and % SDS)less stringent that those described above. An example of moderatelystringent conditions is overnight incubation at 37° C. in a solutioncomprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextransulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed bywashing the filters in 1×SSC at about 37-50° C. The skilled artisan willrecognize how to adjust the temperature, ionic strength, etc. asnecessary to accommodate factors such as probe length and the like.

Antibody “effector functions” refer to those biological activitiesattributable to the Fc region (a native sequence Fc region or amino acidsequence variant Fc region) of an antibody, and vary with the antibodyisotype. Examples of antibody effector functions include: C1q bindingand complement dependent cytotoxicity; Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor); and B cellactivation.

“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to aform of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs)present on certain cytotoxic cells (e.g., Natural Killer (NK) cells,neutrophils, and macrophages) enable these cytotoxic effector cells tobind specifically to an antigen-bearing target cell and subsequentlykill the target cell with cytotoxins. The antibodies “arm” the cytotoxiccells and are absolutely required for such killing. The primary cellsfor mediating ADCC, NK cells, express FcγRIII only, whereas monocytesexpress FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoieticcells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu.Rev. Immunol. 9:457-92 (1991). To assess ADCC activity of a molecule ofinterest, an in vitro ADCC assay, such as that described in U.S. Pat.No. 5,500,362 or 5,821,337 can be performed. Useful effector cells forsuch assays include peripheral blood mononuclear cells (PBMC) andNatural Killer (NK) cells. Alternatively, or additionally, ADCC activityof the molecule of interest can be assessed in vivo, e.g., in a animalmodel such as that disclosed in Clynes et al. (Proc. Natl. Acad. Sci.USA) 95:652-656 (1998).

“Fc receptor” or “FcR” describes a receptor that binds to the Fc regionof an antibody. The preferred FcR is a native sequence human FcR.Moreover, a preferred FcR is one which binds an IgG antibody (a gammareceptor) and includes receptors of the FcγRI, FcγRII, and FcγRIIIsubclasses, including allelic variants and alternatively spliced formsof these receptors. FcγRII receptors include FcγRIIA (an “activatingreceptor”) and FcγRIIB (an “inhibiting receptor”), which have similaramino acid sequences that differ primarily in the cytoplasmic domainsthereof. Activating receptor FcγRIIA contains an immunoreceptortyrosine-based activation motif (ITAM) in its cytoplasmic domainInhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-basedinhibition motif (ITIM) in its cytoplasmic domain (see review M. inDaëron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed inRavetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991); Capel et al.,Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med.126:330-41 (1995). Other FcRs, including those to be identified in thefuture, are encompassed by the term “FcR” herein. The term also includesthe neonatal receptor, FcRn, which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) andKim et al., J. Immunol. 24:249 (1994)).

“Human effector cells” are leukocytes which express one or more FcRs andperform effector functions. Preferably, the cells express at leastFcγRIII and perform ADCC effector function. Examples of human leukocyteswhich mediate ADCC include peripheral blood mononuclear cells (PBMC),natural killer (NK) cells, monocytes, cytotoxic T cells, andneutrophils; with PBMCs and NK cells being preferred. The effector cellscan be isolated from a native source, e.g., from blood.

“Complement dependent cytotoxicity” or “CDC” refers to the lysis of atarget cell in the presence of complement. Activation of the classicalcomplement pathway is initiated by the binding of the first component ofthe complement system (C1q) to antibodies (of the appropriate subclass)which are bound to their cognate antigen. To assess complementactivation, a CDC assay, e.g., as described in Gazzano-Santoro et al.,J. Immunol. Methods 202:163 (1996), can be performed.

The term “therapeutically effective amount” refers to an amount of anantibody or antibody fragment to treat a disease or disorder in asubject. In the case of tumor (e.g., a cancerous tumor), thetherapeutically effective amount of the antibody or antibody fragment(e.g., a multispecific antibody or antibody fragment that specificallybinds HER2 and VEGF) may reduce the number of cancer cells; reduce theprimary tumor size; inhibit (i.e., slow to some extent and preferablystop) cancer cell infiltration into peripheral organs; inhibit (i.e.,slow to some extent and preferably stop) tumor metastasis; inhibit, tosome extent, tumor growth; and/or relieve to some extent one or more ofthe symptoms associated with the disorder. To the extent the antibody orantibody fragment may prevent growth and/or kill existing cancer cells,it may be cytostatic and/or cytotoxic. For cancer therapy, efficacy invivo can, for example, be measured by assessing the duration ofsurvival, time to disease progression (TTP), the response rates (RR),duration of response, and/or quality of life.

By “reduce or inhibit” is meant the ability to cause an overall decreasepreferably of 20% or greater, more preferably of 50% or greater, andmost preferably of 75%, 85%, 90%, 95%, or greater. Reduce or inhibit canrefer to the symptoms of the disorder being treated, the presence orsize of metastases, the size of the primary tumor, or the size or numberof the blood vessels in angiogenic disorders.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth/proliferation. Included in this definition arebenign and malignant cancers. Examples of cancer include but are notlimited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Moreparticular examples of such cancers include squamous cell cancer,small-cell lung cancer, non-small cell lung cancer, adenocarcinoma ofthe lung, squamous carcinoma of the lung, cancer of the peritoneum,hepatocellular cancer, gastric or stomach cancer includinggastrointestinal cancer, pancreatic cancer, glioblastoma, glioma,cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma,breast cancer, colon cancer, colorectal cancer, endometrial or uterinecarcinoma, salivary gland carcinoma, kidney cancer (e.g., renal cellcarcinoma), liver cancer, prostate cancer, vulval cancer, thyroidcancer, hepatic carcinoma, anal carcinoma, penile carcinoma, melanoma,and various types of head and neck cancer.

By “early stage cancer” is meant a cancer that is not invasive ormetastatic or is classified as a Stage 0, I, or II cancer.

The term “precancerous” refers to a condition or a growth that typicallyprecedes or develops into a cancer.

By “non-metastatic” is meant a cancer that is benign or that remains atthe primary site and has not penetrated into the lymphatic or bloodvessel system or to tissues other than the primary site. Generally, anon-metastatic cancer is any cancer that is a Stage 0, I, or II cancer,and occasionally a Stage III cancer.

A “non-malignant disease or disorder involving abnormal activation ofHER2” is a condition which does not involve a cancer where abnormalactivation of HER2 is occurring in cells or tissue of the subjecthaving, or predisposed to, the disease or disorder. Examples of suchdiseases or disorders include autoimmune disease (e.g. psoriasis), seedefinition below; endometriosis; scleroderma; restenosis; polyps such ascolon polyps, nasal polyps or gastrointestinal polyps; fibroadenoma;respiratory disease (e.g., chronic bronchitis, asthma including acuteasthma and allergic asthma, cystic fibrosis, bronchiectasis, allergic orother rhinitis or sinusitis, α1-anti-trypsin deficiency, coughs,pulmonary emphysema, pulmonary fibrosis or hyper-reactive airways,chronic obstructive pulmonary disease, and chronic obstructive lungdisorder); cholecystitis; neurofibromatosis; polycystic kidney disease;inflammatory diseases; skin disorders including psoriasis anddermatitis; vascular disease; conditions involving abnormalproliferation of vascular epithelial cells; gastrointestinal ulcers;Menetrier's disease, secreting adenomas or protein loss syndrome; renaldisorders; angiogenic disorders; ocular disease such as age relatedmacular degeneration, presumed ocular histoplasmosis syndrome, retinalneovascularization from proliferative diabetic retinopathy, retinalvascularization, diabetic retinopathy, or age related maculardegeneration; bone associated pathologies such as osteoarthritis,rickets and osteoporosis; damage following a cerebral ischemic event;fibrotic or edemia diseases such as hepatic cirrhosis, lung fibrosis,carcoidosis, throiditis, hyperviscosity syndrome systemic, OslerWeber-Rendu disease, chronic occlusive pulmonary disease, or edemafollowing burns, trauma, radiation, stroke, hypoxia or ischemia;hypersensitivity reaction of the skin; diabetic retinopathy and diabeticnephropathy; Guillain-Barre syndrome; graft versus host disease ortransplant rejection; Paget's disease; bone or joint inflammation;photoaging (e.g. caused by UV radiation of human skin); benign prostatichypertrophy; certain microbial infections including microbial pathogensselected from adenovirus, hantaviruses, Borrelia burgdorferi, Yersiniaspp. and Bordetella pertussis; thrombus caused by platelet aggregation;reproductive conditions such as endometriosis, ovarian hyperstimulationsyndrome, preeclampsia, dysfunctional uterine bleeding, ormenometrorrhagia; synovitis; atheroma; acute and chronic nephropathies(including proliferative glomerulonephritis and diabetes-induced renaldisease); eczema; hypertrophic scar formation; endotoxic shock andfungal infection; familial adenomatosis polyposis; neurodedenerativediseases (e.g. Alzheimer's disease, AIDS-related dementia, Parkinson'sdisease, amyotrophic lateral sclerosis, retinitis pigmentosa, spinalmuscular atrophy and cerebellar degeneration); myelodysplasticsyndromes; aplastic anemia; ischemic injury; fibrosis of the lung,kidney or liver; T-cell mediated hypersensitivity disease; infantilehypertrophic pyloric stenosis; urinary obstructive syndrome; psoriaticarthritis; and Hasimoto's thyroiditis.

An “autoimmune disease” herein is a disease or disorder arising from anddirected against an individual's own tissues or a co-segregate ormanifestation thereof or resulting condition therefrom. Examples ofautoimmune diseases or disorders include, but are not limited toarthritis (rheumatoid arthritis such as acute arthritis, chronicrheumatoid arthritis, gouty arthritis, acute gouty arthritis, chronicinflammatory arthritis, degenerative arthritis, infectious arthritis,Lyme arthritis, proliferative arthritis, psoriatic arthritis, vertebralarthritis, and juvenile-onset rheumatoid arthritis, osteoarthritis,arthritis chronica progrediente, arthritis deformans, polyarthritischronica primaria, reactive arthritis, and ankylosing spondylitis),inflammatory hyperproliferative skin diseases, psoriasis such as plaquepsoriasis, gutatte psoriasis, pustular psoriasis, and psoriasis of thenails, dermatitis including contact dermatitis, chronic contactdermatitis, allergic dermatitis, allergic contact dermatitis, dermatitisherpetiformis, and atopic dermatitis, x-linked hyper IgM syndrome,urticaria such as chronic allergic urticaria and chronic idiopathicurticaria, including chronic autoimmune urticaria,polymyositis/dermatomyositis, juvenile dermatomyositis, toxic epidermalnecrolysis, scleroderma (including systemic scleroderma), sclerosis suchas systemic sclerosis, multiple sclerosis (MS) such as spino-optical MS,primary progressive MS (PPMS), and relapsing remitting MS (RRMS),progressive systemic sclerosis, atherosclerosis, arteriosclerosis,sclerosis disseminata, and ataxic sclerosis, inflammatory bowel disease(IBD) (for example, Crohn's disease, autoimmune-mediatedgastrointestinal diseases, colitis such as ulcerative colitis, colitisulcerosa, microscopic colitis, collagenous colitis, colitis polyposa,necrotizing enterocolitis, and transmural colitis, and autoimmuneinflammatory bowel disease), pyoderma gangrenosum, erythema nodosum,primary sclerosing cholangitis, episcleritis), respiratory distresssyndrome, including adult or acute respiratory distress syndrome (ARDS),meningitis, inflammation of all or part of the uvea, iritis,choroiditis, an autoimmune hematological disorder, rheumatoidspondylitis, sudden hearing loss, IgE-mediated diseases such asanaphylaxis and allergic and atopic rhinitis, encephalitis such asRasmussen's encephalitis and limbic and/or brainstem encephalitis,uveitis, such as anterior uveitis, acute anterior uveitis, granulomatousuveitis, nongranulomatous uveitis, phacoantigenic uveitis, posterioruveitis, or autoimmune uveitis, glomerulonephritis (GN) with and withoutnephrotic syndrome such as chronic or acute glomerulonephritis such asprimary GN, immune-mediated GN, membranous GN (membranous nephropathy),idiopathic membranous GN or idiopathic membranous nephropathy, membrano-or membranous proliferative GN (MPGN), including Type I and Type II, andrapidly progressive GN, allergic conditions, allergic reaction, eczemaincluding allergic or atopic eczema, asthma such as asthma bronchiale,bronchial asthma, and autoimmune asthma, conditions involvinginfiltration of T cells and chronic inflammatory responses, chronicpulmonary inflammatory disease, autoimmune myocarditis, leukocyteadhesion deficiency, systemic lupus erythematosus (SLE) or systemiclupus erythematodes such as cutaneous SLE, subacute cutaneous lupuserythematosus, neonatal lupus syndrome (NLE), lupus erythematosusdisseminatus, lupus (including nephritis, cerebritis, pediatric,non-renal, extra-renal, discoid, alopecia), juvenile onset (Type I)diabetes mellitus, including pediatric insulin-dependent diabetesmellitus (IDDM), adult onset diabetes mellitus (Type II diabetes),autoimmune diabetes, idiopathic diabetes insipidus, immune responsesassociated with acute and delayed hypersensitivity mediated by cytokinesand T-lymphocytes, tuberculosis, sarcoidosis, granulomatosis includinglymphomatoid granulomatosis, Wegener's granulomatosis, agranulocytosis,vasculitides, including vasculitis (including large vessel vasculitis(including polymyalgia rheumatica and giant cell (Takayasu's)arteritis), medium vessel vasculitis (including Kawasaki's disease andpolyarteritis nodosa), microscopic polyarteritis, CNS vasculitis,necrotizing, cutaneous, or hypersensitivity vasculitis, systemicnecrotizing vasculitis, and ANCA-associated vasculitis, such asChurg-Strauss vasculitis or syndrome (CSS)), temporal arteritis,aplastic anemia, autoimmune aplastic anemia, Coombs positive anemia,Diamond Blackfan anemia, hemolytic anemia or immune hemolytic anemiaincluding autoimmune hemolytic anemia (AIHA), pernicious anemia (anemiaperniciosa), Addison's disease, pure red cell anemia or aplasia (PRCA),Factor VIII deficiency, hemophilia A, autoimmune neutropenia,pancytopenia, leukopenia, diseases involving leukocyte diapedesis, CNSinflammatory disorders, multiple organ injury syndrome such as thosesecondary to septicemia, trauma or hemorrhage, antigen-antibodycomplex-mediated diseases, anti-glomerular basement membrane disease,anti-phospholipid antibody syndrome, allergic neuritis, Bechet's orBehcet's disease, Castleman's syndrome, Goodpasture's syndrome,Reynaud's syndrome, Sjogren's syndrome, Stevens-Johnson syndrome,pemphigoid such as pemphigoid bullous and skin pemphigoid, pemphigus(including pemphigus vulgaris, pemphigus foliaceus, pemphigusmucus-membrane pemphigoid, and pemphigus erythematosus), autoimmunepolyendocrinopathies, Reiter's disease or syndrome, immune complexnephritis, antibody-mediated nephritis, neuromyelitis optica,polyneuropathies, chronic neuropathy such as IgM polyneuropathies orIgM-mediated neuropathy, thrombocytopenia (as developed by myocardialinfarction patients, for example), including thrombotic thrombocytopenicpurpura (TTP) and autoimmune or immune-mediated thrombocytopenia such asidiopathic thrombocytopenic purpura (ITP) including chronic or acuteITP, autoimmune disease of the testis and ovary including autoimmuneorchitis and oophoritis, primary hypothyroidism, hypoparathyroidism,autoimmune endocrine diseases including thyroiditis such as autoimmunethyroiditis, Hashimoto's disease, chronic thyroiditis (Hashimoto'sthyroiditis), or subacute thyroiditis, autoimmune thyroid disease,idiopathic hypothyroidism, Grave's disease, polyglandular syndromes suchas autoimmune polyglandular syndromes (or polyglandular endocrinopathysyndromes), paraneoplastic syndromes, including neurologicparaneoplastic syndromes such as Lambert-Eaton myasthenic syndrome orEaton-Lambert syndrome, stiff-man or stiff-person syndrome,encephalomyelitis such as allergic encephalomyelitis orencephalomyelitis allergica and experimental allergic encephalomyelitis(EAE), myasthenia gravis such as thymoma-associated myasthenia gravis,cerebellar degeneration, neuromyotonia, opsoclonus or opsoclonusmyoclonus syndrome (OMS), and sensory neuropathy, multifocal motorneuropathy, Sheehan's syndrome, autoimmune hepatitis, chronic hepatitis,lupoid hepatitis, giant cell hepatitis, chronic active hepatitis orautoimmune chronic active hepatitis, lymphoid interstitial pneumonitis,bronchiolitis obliterans (non-transplant) vs NSIP, Guillain-Barrésyndrome, Berger's disease (IgA nephropathy), idiopathic IgAnephropathy, linear IgA dermatosis, primary biliary cirrhosis,pneumonocirrhosis, autoimmune enteropathy syndrome, Celiac disease,Coeliac disease, celiac sprue (gluten enteropathy), refractory sprue,idiopathic sprue, cryoglobulinemia, amylotrophic lateral sclerosis (ALS;Lou Gehrig's disease), coronary artery disease, autoimmune ear diseasesuch as autoimmune inner ear disease (AIED), autoimmune hearing loss,opsoclonus myoclonus syndrome (OMS), polychondritis such as refractoryor relapsed polychondritis, pulmonary alveolar proteinosis, amyloidosis,scleritis, a non-cancerous lymphocytosis, a primary lymphocytosis, whichincludes monoclonal B cell lymphocytosis (e.g., benign monoclonalgammopathy and monoclonal gammopathy of undetermined significance,MGUS), peripheral neuropathy, paraneoplastic syndrome, channelopathiessuch as epilepsy, migraine, arrhythmia, muscular disorders, deafness,blindness, periodic paralysis, and channelopathies of the CNS, autism,inflammatory myopathy, focal segmental glomerulosclerosis (FSGS),endocrine ophthalmopathy, uveoretinitis, chorioretinitis, autoimmunehepatological disorder, fibromyalgia, multiple endocrine failure,Schmidt's syndrome, adrenalitis, gastric atrophy, presenile dementia,demyelinating diseases such as autoimmune demyelinating diseases,diabetic nephropathy, Dressler's syndrome, alopecia areata, CRESTsyndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility,sclerodactyly, and telangiectasia), male and female autoimmuneinfertility, mixed connective tissue disease, Chagas' disease, rheumaticfever, recurrent abortion, farmer's lung, erythema multiforme,post-cardiotomy syndrome, Cushing's syndrome, bird-fancier's lung,allergic granulomatous angiitis, benign lymphocytic angiitis, Alport'ssyndrome, alveolitis such as allergic alveolitis and fibrosingalveolitis, interstitial lung disease, transfusion reaction, leprosy,malaria, leishmaniasis, kypanosomiasis, schistosomiasis, ascariasis,aspergillosis, Sampter's syndrome, Caplan's syndrome, dengue,endocarditis, endomyocardial fibrosis, diffuse interstitial pulmonaryfibrosis, interstitial lung fibrosis, idiopathic pulmonary fibrosis,cystic fibrosis, endophthalmitis, erythema elevatum et diutinum,erythroblastosis fetalis, eosinophilic faciitis, Shulman's syndrome,Felty's syndrome, flariasis, cyclitis such as chronic cyclitis,heterochronic cyclitis, iridocyclitis, or Fuch's cyclitis,Henoch-Schonlein purpura, human immunodeficiency virus (HIV) infection,echovirus infection, cardiomyopathy, Alzheimer's disease, parvovirusinfection, rubella virus infection, post-vaccination syndromes,congenital rubella infection, Epstein-Barr virus infection, mumps,Evan's syndrome, autoimmune gonadal failure, Sydenham's chorea,post-streptococcal nephritis, thromboangitis ubiterans, thyrotoxicosis,tabes dorsalis, chorioiditis, giant cell polymyalgia, endocrineophthamopathy, chronic hypersensitivity pneumonitis,keratoconjunctivitis sicca, epidemic keratoconjunctivitis, idiopathicnephritic syndrome, minimal change nephropathy, benign familial andischemia-reperfusion injury, retinal autoimmunity, joint inflammation,bronchitis, chronic obstructive airway disease, silicosis, aphthae,aphthous stomatitis, arteriosclerotic disorders, aspermiogenese,autoimmune hemolysis, Boeck's disease, cryoglobulinemia, Dupuytren'scontracture, endophthalmia phacoanaphylactica, enteritis allergica,erythema nodosum leprosum, idiopathic facial paralysis, chronic fatiguesyndrome, febris rheumatica, Hamman-Rich's disease, sensoneural hearingloss, haemoglobinuria paroxysmatica, hypogonadism, ileitis regionalis,leucopenia, mononucleosis infectiosa, traverse myelitis, primaryidiopathic myxedema, nephrosis, ophthalmia symphatica, orchitisgranulomatosa, pancreatitis, polyradiculitis acuta, pyodermagangrenosum, Quervain's thyreoiditis, acquired spenic atrophy,infertility due to antispermatozoan antibodies, non-malignant thymoma,vitiligo, SCID and Epstein-Barr virus-associated diseases, acquiredimmune deficiency syndrome (AIDS), parasitic diseases such asLeishmania, toxic-shock syndrome, food poisoning, conditions involvinginfiltration of T cells, leukocyte-adhesion deficiency, immune responsesassociated with acute and delayed hypersensitivity mediated by cytokinesand T-lymphocytes, diseases involving leukocyte diapedesis, multipleorgan injury syndrome, antigen-antibody complex-mediated diseases,antiglomerular basement membrane disease, allergic neuritis, autoimmunepolyendocrinopathies, oophoritis, primary myxedema, autoimmune atrophicgastritis, sympathetic ophthalmia, rheumatic diseases, mixed connectivetissue disease, nephrotic syndrome, insulitis, polyendocrine failure,peripheral neuropathy, autoimmune polyglandular syndrome type I,adult-onset idiopathic hypoparathyroidism (AOIH), alopecia totalis,dilated cardiomyopathy, epidermolisis bullosa acquisita (EBA),hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosingcholangitis, purulent or nonpurulent sinusitis, acute or chronicsinusitis, ethmoid, frontal, maxillary, or sphenoid sinusitis, aneosinophil-related disorder such as eosinophilia, pulmonary infiltrationeosinophilia, eosinophilia-myalgia syndrome, Loffler's syndrome, chroniceosinophilic pneumonia, tropical pulmonary eosinophilia,bronchopneumonic aspergillosis, aspergilloma, or granulomas containingeosinophils, anaphylaxis, seronegative spondyloarthritides,polyendocrine autoimmune disease, sclerosing cholangitis, sclera,episclera, chronic mucocutaneous candidiasis, Bruton's syndrome,transient hypogammaglobulinemia of infancy, Wiskott-Aldrich syndrome,ataxia telangiectasia, autoimmune disorders associated with collagendisease, rheumatism, neurological disease, ischemic re-perfusiondisorder, reduction in blood pressure response, vascular dysfunction,antgiectasis, tissue injury, cardiovascular ischemia, hyperalgesia,cerebral ischemia, and disease accompanying vascularization, allergichypersensitivity disorders, glomerulonephritides, reperfusion injury,reperfusion injury of myocardial or other tissues, dermatoses with acuteinflammatory components, acute purulent meningitis or other centralnervous system inflammatory disorders, ocular and orbital inflammatorydisorders, granulocyte transfusion-associated syndromes,cytokine-induced toxicity, acute serious inflammation, chronicintractable inflammation, pyelitis, pneumonocirrhosis, diabeticretinopathy, diabetic large-artery disorder, endarterial hyperplasia,peptic ulcer, valvulitis, and endometriosis.

An “anti-angiogenesis agent” or “angiogenesis inhibitor” refers to asmall molecular weight substance, a polynucleotide, a polypeptide, anisolated protein, a recombinant protein, an antibody, or conjugates orfusion proteins thereof, that inhibits angiogenesis, vasculogenesis, orundesirable vascular permeability, either directly or indirectly. Forexample, an anti-angiogenesis agent is an antibody or other antagonistto an angiogenic agent as defined above, e.g., antibodies to VEGF (e.g.,bevacizumab (AVASTIN®), bH1, bH1-44, bH1-81), antibodies to VEGFreceptors, small molecules that block VEGF receptor signaling (e.g.,PTK787/ZK2284, SU6668, SUTENT/SU11248 (sunitinib malate), AMG706).Anti-angiogensis agents also include native angiogenesis inhibitors,e.g., angiostatin, endostatin, etc. See, e.g., Klagsbrun and D'Amore,Annu. Rev. Physiol., 53:217-39 (1991); Streit and Detmar, Oncogene,22:3172-3179 (2003) (e.g., Table 3 listing anti-angiogenic therapy inmalignant melanoma); Ferrara & Alitalo, Nature Medicine 5(12):1359-1364(1999); Tonini et al., Oncogene, 22:6549-6556 (2003) (e.g., Table 2listing anti-angiogenic factors); and, Sato Int. J. Clin. Oncol.,8:200-206 (2003) (e.g., Table 1 lists anti-angiogenic agents used inclinical trials). Dysregulation of angiogenesis can lead to manydisorders that can be treated by compositions and methods of theinvention. These disorders include both non-neoplastic and neoplasticconditions.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of a cell and/or causes destruction ofa cell. The term is intended to include radioactive isotopes (e.g.,At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², Ra²²³, P³², andradioactive isotopes of Lu), chemotherapeutic agents, e.g.,methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine,etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil,daunorubicin or other intercalating agents, enzymes and fragmentsthereof such as nucleolytic enzymes, antibiotics, and toxins such assmall molecule toxins or enzymatically active toxins of bacterial,fungal, plant or animal origin, including fragments and/or variantsthereof, and the various antitumor or anticancer agents disclosedherein. Other cytotoxic agents are described herein. A tumoricidal agentcauses destruction of tumor cells.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines suchas benzodopa, carboquone, meturedopa, and uredopa; ethylenimines andmethylamelamines including altretamine, triethylenemelamine,trietylenephosphoramide, triethiylenethiophosphoramide andtrimethylolomelamine; acetogenins (especially bullatacin andbullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®);beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin(including the synthetic analogue topotecan (HYCAMTIN®), CPT-11(irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including itsadozelesin, carzelesin and bizelesin synthetic analogues);podophyllotoxin; podophyllinic acid; teniposide; cryptophycins(particularly cryptophycin 1 and cryptophycin 8); dolastatin;duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1);eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine;antibiotics such as the enediyne antibiotics (e.g., calicheamicin,especially calicheamicin gamma 1 (see, e.g., Agnew, Chem Intl. Ed.Engl., 33: 183-186 (1994)); dynemicin, including dynemicin A; anesperamicin; as well as neocarzinostatin chromophore and relatedchromoprotein enediyne antibiotic chromophores), aclacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN®doxorubicin (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS NaturalProducts, Eugene, Oreg.); razoxane; rhizoxin; sizofiran; spirogermanium;tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine;trichothecenes (especially T-2 toxin, verracurin A, roridin A andanguidine); urethan; vindesine (ELDISINE®, FILDESIN®); dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); thiotepa; taxoids, e.g., TAXOL® paclitaxel(Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™Cremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil;gemcitabine (GEMZAR®); 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine(VELBAN®); platinum; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine (ONCOVIN®); oxaliplatin; leucovovin; vinorelbine(NAVELBINE®); novantrone; edatrexate; daunomycin; aminopterin;ibandronate; topoisomerase inhibitor RFS 2000; difluorometlhylornithine(DMFO); retinoids such as retinoic acid; capecitabine (XELODA®);pharmaceutically acceptable salts, acids or derivatives of any of theabove; as well as combinations of two or more of the above such as CHOP,an abbreviation for a combined therapy of cyclophosphamide, doxorubicin,vincristine, and prednisolone, and FOLFOX, an abbreviation for atreatment regimen with oxaliplatin (ELOXATIN™) combined with 5-FU andleucovovin.

Also included in this definition are anti-hormonal agents that act toregulate, reduce, block, or inhibit the effects of hormones that canpromote the growth of cancer, and are often in the form of systemic, orwhole-body treatment. They may be hormones themselves. Examples includeanti-estrogens and selective estrogen receptor modulators (SERMs),including, for example, tamoxifen (including NOLVADEX® tamoxifen),EVISTA® raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene,keoxifene, LY117018, onapristone, and FARESTON® toremifene;anti-progesterones; estrogen receptor down-regulators (ERDs); agentsthat function to suppress or shut down the ovaries, for example,leutinizing hormone-releasing hormone (LHRH) agonists such as LUPRON®and ELIGARD® leuprolide acetate, goserelin acetate, buserelin acetateand tripterelin; other anti-androgens such as flutamide, nilutamide andbicalutamide; and aromatase inhibitors that inhibit the enzymearomatase, which regulates estrogen production in the adrenal glands,such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE®megestrol acetate, AROMASIN® exemestane, formestanie, fadrozole,RIVISOR® vorozole, FEMARA® letrozole, and ARIMIDEX® anastrozole. Inaddition, such definition of chemotherapeutic agents includesbisphosphonates such as clodronate (for example, BONEFOS® or OSTAC®),DIDROCAL® etidronate, NE-58095, ZOMETA® zoledronic acid/zoledronate,FOSAMAX® alendronate, AREDIA® pamidronate, SKELID® tiludronate, orACTONEL® risedronate; as well as troxacitabine (a 1,3-dioxolanenucleoside cytosine analog); antisense oligonucleotides, particularlythose that inhibit expression of genes in signaling pathways implicatedin abherant cell proliferation, such as, for example, PKC-alpha, Raf,H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such asTHERATOPE® vaccine and gene therapy vaccines, for example, ALLOVECTIN®vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; LURTOTECAN®topoisomerase 1 inhibitor; ABARELIX® rmRH; lapatinib ditosylate (anErbB-2 and EGFR dual tyrosine kinase small-molecule inhibitor also knownas GW572016); and pharmaceutically acceptable salts, acids orderivatives of any of the above.

A “growth inhibitory agent” when used herein refers to a compound orcomposition which inhibits growth of a cell either in vitro or in vivo.Thus, the growth inhibitory agent may be one which significantly reducesthe percentage of cells in S phase. Examples of growth inhibitory agentsinclude agents that block cell cycle progression (at a place other thanS phase), such as agents that induce G1 arrest and M-phase arrest.Classical M-phase blockers include the vincas (e.g., vincristine andvinblastine), taxanes, and topoisomerase II inhibitors such asdoxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Theagents that arrest G1 also spill over into S-phase arrest, for example,DNA alkylating agents such as tamoxifen, prednisone, dacarbazine,mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C.Further information can be found in The Molecular Basis of Cancer,Mendelsohn and Israel, eds., Chapter 1, entitled “Cell cycle regulation,oncogenes, and antineoplastic drugs” by Murakami et al. (WB Saunders:Philadelphia, 1995), especially p. 13. The taxanes (paclitaxel anddocetaxel) are anticancer drugs both derived from the yew tree.Docetaxel (TAXOTERE®, Rhone-Poulenc Rorer), derived from the Europeanyew, is a semisynthetic analogue of paclitaxel (TAXOL®, Bristol-MyersSquibb). Paclitaxel and docetaxel promote the assembly of microtubulesfrom tubulin dimers and stabilize microtubules by preventingdepolymerization, which results in the inhibition of mitosis in cells.

“Anti-cancer therapy” as used herein refers to a treatment that reducesor inhibits cancer in a subject. Examples of anti-cancer therapy includecytotoxic radiotherapy as well as the administration of atherapeutically effective amount of a cytotoxic agent, achemotherapeutic agent, a growth inhibitory agent, a cancer vaccine, anangiogenesis inhibitor, a prodrug, a cytokine, a cytokine antagonist, acorticosteroid, an immunosuppressive agent, an anti-emetic, an antibodyor antibody fragment, or an analgesic to the subject.

The term “prodrug” as used in this application refers to a precursor orderivative form of a pharmaceutically active substance that is lesscytotoxic to tumor cells compared to the parent drug and is capable ofbeing enzymatically activated or converted into the more active parentform. See, e.g., Wilman, “Prodrugs in Cancer Chemotherapy” BiochemicalSociety Transactions, 14, pp. 375-382, 615th Meeting Belfast (1986) andStella et al., “Prodrugs: A Chemical Approach to Targeted DrugDelivery,” Directed Drug Delivery, Borchardt et al., (ed.), pp. 247-267,Humana Press (1985). Prodrugs include, but are not limited to,phosphate-containing prodrugs, thiophosphate-containing prodrugs,sulfate-containing prodrugs, peptide-containing prodrugs, D-aminoacid-modified prodrugs, glycosylated prodrugs, beta-lactam-containingprodrugs, optionally substituted phenoxyacetamide-containing prodrugs oroptionally substituted phenylacetamide-containing prodrugs,5-fluorocytosine and other 5-fluorouridine prodrugs which can beconverted into the more active cytotoxic free drug. Examples ofcytotoxic drugs that can be derivatized into a prodrug form for use inthis invention include, but are not limited to, those chemotherapeuticagents described above.

The term “cytokine” is a generic term for proteins released by one cellpopulation which act on another cell as intercellular mediators.Examples of such cytokines are lymphokines, monokines, and traditionalpolypeptide hormones. Included among the cytokines are growth hormonesuch as human growth hormone (HGH), N-methionyl human growth hormone,and bovine growth hormone; parathyroid hormone; thyroxine; insulin;proinsulin; relaxin; prorelaxin; glycoprotein hormones such as folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH), andluteinizing hormone (LH); epidermal growth factor (EGF); hepatic growthfactor; fibroblast growth factor (FGF); prolactin; placental lactogen;tumor necrosis factor-alpha and -beta; mullerian-inhibiting substance;mouse gonadotropin-associated peptide; inhibin; activin; vascularendothelial growth factor; integrin; thrombopoietin (TPO); nerve growthfactors such as NGF-alpha; platelet-growth factor; transforming growthfactors (TGFs) such as TGF-alpha and TGF-beta; insulin-like growthfactor-I and -II; erythropoietin (EPO); osteoinductive factors;interferons such as interferon-alpha, -beta and -gamma colonystimulating factors (CSFs) such as macrophage-CSF (M-CSF);granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);interleukins (ILs) such as IL-1, IL-1alpha, IL-2, IL-3, IL-4, IL-5,IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; a tumor necrosis factorsuch as TNF-alpha or TNF-beta; and other polypeptide factors includingLIF and kit ligand (KL). As used herein, the term cytokine includesproteins from natural sources or from recombinant cell culture andbiologically active equivalents of the native sequence cytokines.

By “cytokine antagonist” is meant a molecule that partially or fullyblocks, inhibits, or neutralizes a biological activity of at least onecytokine. For example, the cytokine antagonists may inhibit cytokineactivity by inhibiting cytokine expression and/or secretion, or bybinding to a cytokine or to a cytokine receptor. Cytokine antagonistsinclude antibodies, synthetic or native-sequence peptides,immunoadhesins, and small-molecule antagonists that bind to a cytokineor cytokine receptor. The cytokine antagonist is optionally conjugatedwith or fused to a cytotoxic agent. Exemplary TNF antagonists areetanercept (ENBREL®), infliximab (REMICADE®), and adalimumab (HUMIRA™).

The term “immunosuppressive agent” as used herein refers to substancesthat act to suppress or mask the immune system of the subject beingtreated. This includes substances that suppress cytokine production,downregulate or suppress self-antigen expression, or mask the MHCantigens. Examples of immunosuppressive agents include2-amino-6-aryl-5-substituted pyrimidines (see U.S. Pat. No. 4,665,077);mycophenolate mofetil such as CELLCEPT®; azathioprine (IMURAN®,AZASAN®/6-mercaptopurine; bromocryptine; danazol; dapsone;glutaraldehyde (which masks the MHC antigens, as described in U.S. Pat.No. 4,120,649); anti-idiotypic antibodies for MHC antigens and MHCfragments; cyclosporin A; steroids such as corticosteroids andglucocorticosteroids, e.g., prednisone, prednisolone such as PEDIAPRED®(prednisolone sodium phosphate) or ORAPRED® (prednisolone sodiumphosphate oral solution), methylprednisolone, and dexamethasone;methotrexate (oral or subcutaneous) (RHEUMATREX®, TREXALL™);hydroxycloroquine/chloroquine; sulfasalazine; leflunomide; cytokine orcytokine receptor antagonists including anti-interferon-γ, -β, or -αantibodies, anti-tumor necrosis factor-α antibodies (infliximab oradalimumab), anti-TNFα immunoadhesin (ENBREL®, etanercept), anti-tumornecrosis factor-β antibodies, anti-interleukin-2 antibodies andanti-IL-2 receptor antibodies; anti-LFA-1 antibodies, includinganti-CD11a and anti-CD18 antibodies; anti-L3T4 antibodies; heterologousanti-lymphocyte globulin; polyclonal or pan-T antibodies, or monoclonalanti-CD3 or anti-CD4/CD4a antibodies; soluble peptide containing a LFA-3binding domain (WO 1990/08187, published Jul. 26, 1990); streptokinase;TGF-β; streptodornase; RNA or DNA from the host; FK506; RS-61443;deoxyspergualin; rapamycin; T-cell receptor (Cohen et al., U.S. Pat. No.5,114,721); T-cell receptor fragments (Offner et al. Science, 251:430-432 (1991); WO 1990/11294; Ianeway, Nature, 341: 482 (1989); and WO1991/01133); T cell receptor antibodies (EP 340,109) such as T10B9;cyclophosphamide (CYTOXAN®); dapsone; penicillamine (CUPRIMINE®); plasmaexchange; or intravenous immunoglobulin (IVIG). These may be used aloneor in combination with each other, particularly combinations of steroidand another immunosuppressive agent or such combinations followed by amaintenance dose with a non-steroid agent to reduce the need forsteroids.

An “analgesic” refers to a drug that acts to inhibit or suppress pain ina subject. Exemplary analgesics include non-steroidal anti-inflammatorydrugs (NSAIDs) including ibuprofen (MOTRIN®), naproxen (NAPROSYN®),acetylsalicylic acid, indomethacin, sulindac, and tolmetin, includingsalts and derivatives thereof, as well as various other medications usedto reduce the stabbing pains that may occur, including anticonvulsants(gabapentin, phenyloin, carbamazepine) or tricyclic antidepressants.Specific examples include acetaminophen, aspirin, amitriptyline(ELAVIL®), carbamazepine (TEGRETOL®), phenyltoin (DILANTIN®), gabapentin(NEURONTIN®), (E)-N-Vanillyl-8-methyl-6-noneamid (CAPSAICIN®), or anerve blocker.

“Corticosteroid” refers to any one of several synthetic or naturallyoccurring substances with the general chemical structure of steroidsthat mimic or augment the effects of the naturally occurringcorticosteroids. Examples of synthetic corticosteroids includeprednisone, prednisolone (including methylprednisolone), dexamethasonetriamcinolone, and betamethasone.

A “cancer vaccine,” as used herein is a composition that stimulates animmune response in a subject against a cancer. Cancer vaccines typicallyconsist of a source of cancer-associated material or cells (antigen)that may be autologous (from self) or allogenic (from others) to thesubject, along with other components (e.g., adjuvants) to furtherstimulate and boost the immune response against the antigen. Cancervaccines desirably result in stimulating the immune system of thesubject to produce antibodies to one or several specific antigens,and/or to produce killer T cells to attack cancer cells that have thoseantigens.

“Cytotoxic radiotherapy” as used herein refers to radiation therapy thatinhibits or prevents the function of cells and/or causes destruction ofcells. Radiation therapy may include, for example, external beamirradiation or therapy with a radioactive labeled agent, such as anantibody. The term is intended to include use of radioactive isotopes(e.g., At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², Ra²²³, P³²,and radioactive isotopes of Lu).

An “anti-emetic” is a compound that reduces or prevents nausea in asubject. Anti-emetic compounds include, for example, neurokinin-1receptor antagonists, 5HT3 receptor antagonists (such as ondansetron,granisetron, tropisetron, and zatisetron), GABAB receptor agonists, suchas baclofen, a corticosteroid such as dexamethasone, KENALOG®,ARISTOCORT®, or NASALIDE®, an antidopaminergic, phenothiazines (forexample prochlorperazine, fluphenazine, thioridazine and mesoridazine),dronabinol, metroclopramide, domperidone, haloperidol, cyclizine,lorazepam, prochlorperazine, and levomepromazine

A “subject” is a vertebrate, preferably a mammal, more preferably ahuman. Mammals include, but are not limited to, farm animals (such ascows), sport animals, pets (such as cats, dogs and horses), primates,mice, and rats.

Commercially available reagents referred to in the Examples were usedaccording to manufacturer's instructions unless otherwise indicated. Thesource of those cells identified in the following Examples, andthroughout the specification, by ATCC accession numbers is the AmericanType Culture Collection, Manassas, Va. Unless otherwise noted, thepresent invention uses standard procedures of recombinant DNAtechnology, such as those described hereinabove and in the followingtextbooks: Sambrook et al., supra; Ausubel et al., Current Protocols inMolecular Biology (Green Publishing Associates and Wiley Interscience,N.Y., 1989); Innis et al., PCR Protocols: A Guide to Methods andApplications (Academic Press, Inc.: N.Y., 1990); Harlow et al.,Antibodies: A Laboratory Manual (Cold Spring Harbor Press: Cold SpringHarbor, 1988); Gait, Oligonucleotide Synthesis (IRL Press: Oxford,1984); Freshney, Animal Cell Culture, 1987; Coligan et al., CurrentProtocols in Immunology, 1991.

Throughout this specification and claims, the word “comprise,” orvariations such as “comprises” or “comprising,” will be understood toimply the inclusion of a stated integer or group of integers but not theexclusion of any other integer or group of integers.

II. Vectors, Host Cells, and Recombinant Methods

For recombinant production of an antibody of the invention, the nucleicacid encoding it is isolated and inserted into a replicable vector forfurther cloning (amplification of the DNA) or for expression. DNAencoding the antibody is readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of the antibody). Many vectors are available. The choice ofvector depends in part on the host cell to be used. Generally, preferredhost cells are of either prokaryotic or eukaryotic (generally mammalian)origin. It will be appreciated that constant regions of any isotype canbe used for this purpose, including IgG, IgM, IgA, IgD, and IgE constantregions, and that such constant regions can be obtained from any humanor animal species.

a. Generating Antibodies Using Prokaryotic Host Cells:

i. Vector Construction

Polynucleotide sequences encoding polypeptide components of the antibodyof the invention can be obtained using standard recombinant techniques.Desired polynucleotide sequences may be isolated and sequenced fromantibody producing cells such as hybridoma cells. Alternatively,polynucleotides can be synthesized using nucleotide synthesizer or PCRtechniques. Once obtained, sequences encoding the polypeptides areinserted into a recombinant vector capable of replicating and expressingheterologous polynucleotides in prokaryotic hosts. Many vectors that areavailable and known in the art can be used for the purpose of thepresent invention. Selection of an appropriate vector will depend mainlyon the size of the nucleic acids to be inserted into the vector and theparticular host cell to be transformed with the vector. Each vectorcontains various components, depending on its function (amplification orexpression of heterologous polynucleotide, or both) and itscompatibility with the particular host cell in which it resides. Thevector components generally include, but are not limited to: an originof replication, a selection marker gene, a promoter, a ribosome bindingsite (RBS), a signal sequence, the heterologous nucleic acid insert anda transcription termination sequence.

In general, plasmid vectors containing replicon and control sequenceswhich are derived from species compatible with the host cell are used inconnection with these hosts. The vector ordinarily carries a replicationsite, as well as marking sequences which are capable of providingphenotypic selection in transformed cells. For example, E. coli istypically transformed using pBR322, a plasmid derived from an E. colispecies. pBR322 contains genes encoding ampicillin (Amp) andtetracycline (Tet) resistance and thus provides easy means foridentifying transformed cells. pBR322, its derivatives, or othermicrobial plasmids or bacteriophage may also contain, or be modified tocontain, promoters which can be used by the microbial organism forexpression of endogenous proteins. Examples of pBR322 derivatives usedfor expression of particular antibodies are described in detail inCarter et al., U.S. Pat. No. 5,648,237.

In addition, phage vectors containing replicon and control sequencesthat are compatible with the host microorganism can be used astransforming vectors in connection with these hosts. For example,bacteriophage such as λGEM™-11 may be utilized in making a recombinantvector which can be used to transform susceptible host cells such as E.coli LE392.

The expression vector of the invention may comprise two or morepromoter-cistron pairs, encoding each of the polypeptide components. Apromoter is an untranslated regulatory sequence located upstream (5′) toa cistron that modulates its expression. Prokaryotic promoters typicallyfall into two classes, inducible and constitutive. An inducible promoteris a promoter that initiates increased levels of transcription of thecistron under its control in response to changes in the culturecondition, e.g., the presence or absence of a nutrient or a change intemperature.

A large number of promoters recognized by a variety of potential hostcells are well known. The selected promoter can be operably linked tocistron DNA encoding the light or heavy chain by removing the promoterfrom the source DNA via restriction enzyme digestion and inserting theisolated promoter sequence into the vector of the invention. Both thenative promoter sequence and many heterologous promoters may be used todirect amplification and/or expression of the target genes. In someembodiments, heterologous promoters are utilized, as they generallypermit greater transcription and higher yields of expressed target geneas compared to the native target polypeptide promoter.

Promoters suitable for use with prokaryotic hosts include the PhoApromoter, the β-galactamase and lactose promoter systems, a tryptophan(trp) promoter system and hybrid promoters such as the tac or the trcpromoter. However, other promoters that are functional in bacteria (suchas other known bacterial or phage promoters) are suitable as well. Theirnucleotide sequences have been published, thereby enabling a skilledworker to ligate them to cistrons encoding the target light and heavychains (Siebenlist et al., (1980) Cell 20: 269) using linkers oradaptors to supply any required restriction sites.

In one aspect of the invention, each cistron within the recombinantvector comprises a secretion signal sequence component that directstranslocation of the expressed polypeptides across a membrane. Ingeneral, the signal sequence may be a component of the vector, or it maybe a part of the target polypeptide DNA that is inserted into thevector. The signal sequence selected for the purpose of this inventionshould be one that is recognized and processed (i.e., cleaved by asignal peptidase) by the host cell. For prokaryotic host cells that donot recognize and process the signal sequences native to theheterologous polypeptides, the signal sequence is substituted by aprokaryotic signal sequence selected, for example, from the groupconsisting of the alkaline phosphatase, penicillinase, Ipp, orheat-stable enterotoxin II (STII) leaders, LamB, PhoE, PelB, OmpA, andMBP. In one embodiment of the invention, the signal sequences used inboth cistrons of the expression system are STII signal sequences orvariants thereof.

In another aspect, the production of the immunoglobulins according tothe invention can occur in the cytoplasm of the host cell, and thereforedoes not require the presence of secretion signal sequences within eachcistron. In that regard, immunoglobulin light and heavy chains areexpressed, folded and assembled to form functional immunoglobulinswithin the cytoplasm. Certain host strains (e.g., the E. colitrxB-strains) provide cytoplasm conditions that are favorable fordisulfide bond formation, thereby permitting proper folding and assemblyof expressed protein subunits (Proba and Pluckthun, Gene, 159:203(1995)).

Prokaryotic host cells suitable for expressing antibodies of theinvention include Archaebacteria and Eubacteria, such as Gram-negativeor Gram-positive organisms. Examples of useful bacteria includeEscherichia (e.g., E. coli), Bacilli (e.g., B. subtilis),Enterobacteria, Pseudomonas species (e.g., P. aeruginosa), Salmonellatyphimurium, Serratia marcescans, Klebsiella, Proteus, Shigella,Rhizobia, Vitreoscilla, or Paracoccus. In one embodiment, gram-negativecells are used. In one embodiment, E. coli cells are used as hosts forthe invention. Examples of E. coli strains include strain W3110(Bachmann, Cellular and Molecular Biology, vol. 2 (Washington, D.C.:American Society for Microbiology, 1987), pp. 1190-1219; ATCC DepositNo. 27,325) and derivatives thereof, including strain 33D3 havinggenotype W3110 ΔfhuA (ΔtonA) ptr3 lac Iq lacL8 ΔompTΔ (nmpc-fepE) degP41kanR (U.S. Pat. No. 5,639,635). Other strains and derivatives thereof,such as E. coli 294 (ATCC 31,446), E. coli B, E. coli λ 1776 (ATCC31,537) and E. coli RV308 (ATCC 31,608) are also suitable. Theseexamples are illustrative rather than limiting. Methods for constructingderivatives of any of the above-mentioned bacteria having definedgenotypes are known in the art and described in, for example, Bass etal., Proteins, 8:309-314 (1990). It is generally necessary to select theappropriate bacteria taking into consideration replicability of thereplicon in the cells of a bacterium. For example, E. coli, Serratia, orSalmonella species can be suitably used as the host when well-knownplasmids such as pBR322, pBR325, pACYC177, or pKN410 are used to supplythe replicon. Typically the host cell should secrete minimal amounts ofproteolytic enzymes, and additional protease inhibitors may desirably beincorporated in the cell culture.

ii. Antibody Production

Host cells are transformed with the above-described expression vectorsand cultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences.

Transformation means introducing DNA into the prokaryotic host so thatthe DNA is replicable, either as an extrachromosomal element or bychromosomal integrant. Depending on the host cell used, transformationis done using standard techniques appropriate to such cells. The calciumtreatment employing calcium chloride is generally used for bacterialcells that contain substantial cell-wall barriers. Another method fortransformation employs polyethylene glycol/DMSO. Yet another techniqueused is electroporation.

Prokaryotic cells used to produce the polypeptides of the invention aregrown in media known in the art and suitable for culture of the selectedhost cells. Examples of suitable media include Luria broth (LB) plusnecessary nutrient supplements. In some embodiments, the media alsocontains a selection agent, chosen based on the construction of theexpression vector, to selectively permit growth of prokaryotic cellscontaining the expression vector. For example, ampicillin is added tomedia for growth of cells expressing ampicillin resistant gene.

Any necessary supplements besides carbon, nitrogen, and inorganicphosphate sources may also be included at appropriate concentrationsintroduced alone or as a mixture with another supplement or medium suchas a complex nitrogen source. Optionally the culture medium may containone or more reducing agents selected from the group consisting ofglutathione, cysteine, cystamine, thioglycollate, dithioerythritol anddithiothreitol.

The prokaryotic host cells are cultured at suitable temperatures. For E.coli growth, for example, the preferred temperature ranges from about20° C. to about 39° C., more preferably from about 25° C. to about 37°C., even more preferably at about 30° C. The pH of the medium may be anypH ranging from about 5 to about 9, depending mainly on the hostorganism. For E. coli, the pH is preferably from about 6.8 to about 7.4,and more preferably about 7.0.

If an inducible promoter is used in the expression vector of theinvention, protein expression is induced under conditions suitable forthe activation of the promoter. In one aspect of the invention, PhoApromoters are used for controlling transcription of the polypeptides.Accordingly, the transformed host cells are cultured in aphosphate-limiting medium for induction. Preferably, thephosphate-limiting medium is the C.R.A.P medium (see, e.g., Simmons etal., J. Immunol. Methods (2002), 263:133-147). A variety of otherinducers may be used, according to the vector construct employed, as isknown in the art.

In one embodiment, the expressed polypeptides of the present inventionare secreted into and recovered from the periplasm of the host cells.Protein recovery typically involves disrupting the microorganism,generally by such means as osmotic shock, sonication or lysis. Oncecells are disrupted, cell debris or whole cells may be removed bycentrifugation or filtration. The proteins may be further purified, forexample, by affinity resin chromatography. Alternatively, proteins canbe transported into the culture media and isolated therein. Cells may beremoved from the culture and the culture supernatant being filtered andconcentrated for further purification of the proteins produced. Theexpressed polypeptides can be further isolated and identified usingcommonly known methods such as polyacrylamide gel electrophoresis (PAGE)and Western blot assay.

In one aspect of the invention, antibody production is conducted inlarge quantity by a fermentation process. Various large-scale fed-batchfermentation procedures are available for production of recombinantproteins. Large-scale fermentations have at least 1000 liters ofcapacity, preferably about 1,000 to 100,000 liters of capacity. Thesefermentors use agitator impellers to distribute oxygen and nutrients,especially glucose (the preferred carbon/energy source) Small-scalefermentation refers generally to fermentation in a fermentor that is nomore than approximately 100 liters in volumetric capacity, and can rangefrom about 1 liter to about 100 liters.

In a fermentation process, induction of protein expression is typicallyinitiated after the cells have been grown under suitable conditions to adesired density, e.g., an OD550 of about 180-220, at which stage thecells are in the early stationary phase. A variety of inducers may beused, according to the vector construct employed, as is known in the artand described above. Cells may be grown for shorter periods prior toinduction. Cells are usually induced for about 12-50 hours, althoughlonger or shorter induction time may be used.

To improve the production yield and quality of the polypeptides of theinvention, various fermentation conditions can be modified. For example,to improve the proper assembly and folding of the secreted antibodypolypeptides, additional vectors overexpressing chaperone proteins, suchas Dsb proteins (DsbA, DsbB, DsbC, DsbD, and/or DsbG) or FkpA (apeptidylprolyl cis,trans-isomerase with chaperone activity) can be usedto co-transform the host prokaryotic cells. The chaperone proteins havebeen demonstrated to facilitate the proper folding and solubility ofheterologous proteins produced in bacterial host cells. Chen et al.,(1999) J. Biol. Chem. 274:19601-19605; Georgiou et al., U.S. Pat. No.6,083,715; Georgiou et al., U.S. Pat. No. 6,027,888; Bothmann andPluckthun (2000) J. Biol. Chem. 275:17100-17105; Ramm and Pluckthun,(2000) J. Biol. Chem. 275:17106-17113; Arie et al., (2001) Mol.Microbiol. 39:199-210.

To minimize proteolysis of expressed heterologous proteins (especiallythose that are proteolytically sensitive), certain host strainsdeficient for proteolytic enzymes can be used for the present invention.For example, host cell strains may be modified to effect geneticmutation(s) in the genes encoding known bacterial proteases such asProtease III, OmpT, DegP, Tsp, Protease I, Protease Mi, Protease V,Protease VI, and combinations thereof. Some E. coli protease-deficientstrains are available and described in, for example, Joly et al.,(1998), Proc. Natl. Acad. Sci. USA 95:2773-2777; Georgiou et al., U.S.Pat. No. 5,264,365; Georgiou et al., U.S. Pat. No. 5,508,192; Hara etal., Microbial Drug Resistance, 2:63-72 (1996).

In one embodiment, E. coli strains deficient for proteolytic enzymes andtransformed with plasmids overexpressing one or more chaperone proteinsare used as host cells in the expression system of the invention.

iii. Antibody Purification

Standard protein purification methods known in the art can be employed.The following procedures are exemplary of suitable purificationprocedures: fractionation on immunoaffinity or ion-exchange columns,ethanol precipitation, reverse phase HPLC, chromatography on silica oron a cation-exchange resin such as DEAE, chromatofocusing, SDS-PAGE,ammonium sulfate precipitation, and gel filtration using, for example,Sephadex G-75.

In one aspect, Protein A immobilized on a solid phase is used forimmunoaffinity purification of the full length antibody products of theinvention. Protein A is a 41 kD cell wall protein from Staphylococcusaureus which binds with a high affinity to the Fc region of antibodies.Lindmark et al., (1983) J. Immunol. Meth. 62:1-13. The solid phase towhich Protein A is immobilized is preferably a column comprising a glassor silica surface, more preferably a controlled pore glass column or asilicic acid column. In some applications, the column has been coatedwith a reagent, such as glycerol, in an attempt to prevent nonspecificadherence of contaminants.

As the first step of purification, the preparation derived from the cellculture as described above is applied onto the Protein A immobilizedsolid phase to allow specific binding of the antibody of interest toProtein A. The solid phase is then washed to remove contaminantsnon-specifically bound to the solid phase. Finally the antibody ofinterest is recovered from the solid phase by elution.

b. Generating Antibodies Using Eukaryotic Host Cells:

The vector components generally include, but are not limited to, one ormore of the following: a signal sequence, an origin of replication, oneor more marker genes, an enhancer element, a promoter, and atranscription termination sequence.

(i) Signal Sequence Component

A vector for use in a eukaryotic host cell may also contain a signalsequence or other polypeptide having a specific cleavage site at theN-terminus of the mature protein or polypeptide of interest. Theheterologous signal sequence selected preferably is one that isrecognized and processed (i.e., cleaved by a signal peptidase) by thehost cell. In mammalian cell expression, mammalian signal sequences aswell as viral secretory leaders, for example, the herpes simplex gDsignal, are available.

The DNA for such precursor region is ligated in reading frame to DNAencoding the antibody.

(ii) Origin of Replication

Generally, an origin of replication component is not needed formammalian expression vectors. For example, the SV40 origin may typicallybe used only because it contains the early promoter.

(iii) Selection Gene Component

Expression and cloning vectors may contain a selection gene, also termeda selectable marker. Typical selection genes encode proteins that (a)confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tetracycline, (b) complement auxotrophicdeficiencies, where relevant, or (c) supply critical nutrients notavailable from complex media.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene produce a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin, mycophenolic acid, and hygromycin.

Another example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take up theantibody nucleic acid, such as DHFR, thymidine kinase, metallothionein-Iand -II, preferably primate metallothionein genes, adenosine deaminase,ornithine decarboxylase, etc.

For example, cells transformed with the DHFR selection gene are firstidentified by culturing all of the transformants in a culture mediumthat contains methotrexate (Mtx), a competitive antagonist of DHFR. Anappropriate host cell when wild-type DHFR is employed is the Chinesehamster ovary (CHO) cell line deficient in DHFR activity (e.g., ATCCCRL-9096).

Alternatively, host cells (particularly wild-type hosts that containendogenous DHFR) transformed or co-transformed with DNA sequencesencoding an antibody, wild-type DHFR protein, and another selectablemarker such as aminoglycoside 3′-phosphotransferase (APH) can beselected by cell growth in medium containing a selection agent for theselectable marker such as an aminoglycosidic antibiotic, e.g.,kanamycin, neomycin, or G418. See U.S. Pat. No. 4,965,199.

(iv) Promoter Component

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to the antibodypolypeptide nucleic acid. Promoter sequences are known for eukaryotes.Virtually alleukaryotic genes have an AT-rich region locatedapproximately 25 to 30 bases upstream from the site where transcriptionis initiated. Another sequence found 70 to 80 bases upstream from thestart of transcription of many genes is a CNCAAT region where N may beany nucleotide. At the 3′ end of most eukaryotic genes is an AATAAAsequence that may be the signal for addition of the poly A tail to the3′ end of the coding sequence. All of these sequences are suitablyinserted into eukaryotic expression vectors.

Antibody polypeptide transcription from vectors in mammalian host cellsis controlled, for example, by promoters obtained from the genomes ofviruses such as polyoma virus, fowlpox virus, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, a retrovirus, hepatitis-B virus, and Simian Virus 40(SV40), from heterologous mammalian promoters, e.g., the actin promoteror an immunoglobulin promoter, from heat-shock promoters, provided suchpromoters are compatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. A system for expressing DNA in mammalian hosts using thebovine papilloma virus as a vector is disclosed in U.S. Pat. No.4,419,446. A modification of this system is described in U.S. Pat. No.4,601,978. Alternatively, the Rous Sarcoma Virus long terminal repeatcan be used as the promoter.

(v) Enhancer Element Component

Transcription of DNA encoding the antibody polypeptide of this inventionby higher eukaryotes is often increased by inserting an enhancersequence into the vector. Many enhancer sequences are now known frommammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin).Typically, however, one will use an enhancer from a eukaryotic cellvirus. Examples include the SV40 enhancer on the late side of thereplication origin (bp 100-270), the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers. See also Yaniv, Nature 297:17-18(1982) on enhancing elements for activation of eukaryotic promoters. Theenhancer may be spliced into the vector at a position 5′ or 3′ to theantibody polypeptide-encoding sequence, but is preferably located at asite 5′ from the promoter.

(vi) Transcription Termination Component

Expression vectors used in eukaryotic host cells will typically alsocontain sequences necessary for the termination of transcription and forstabilizing the mRNA. Such sequences are commonly available from the 5′and, occasionally 3′, untranslated regions of eukaryotic or viral DNAsor cDNAs. These regions contain nucleotide segments transcribed aspolyadenylated fragments in the untranslated portion of the mRNAencoding an antibody. One useful transcription termination component isthe bovine growth hormone polyadenylation region. See WO94/11026 and theexpression vector disclosed therein.

(vii) Selection and Transformation of Host Cells

Suitable host cells for cloning or expressing the DNA in the vectorsherein include higher eukaryote cells described herein, includingvertebrate host cells. Propagation of vertebrate cells in culture(tissue culture) has become a routine procedure. Examples of usefulmammalian host cell lines are monkey kidney CV1 line transformed by SV40(COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cellssubcloned for growth in suspension culture, Graham et al., J. Gen.Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10);Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad.Sci. USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol.Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70);African green monkey kidney cells (VERO-76, ATCC CRL-1587); humancervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK,ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); humanlung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065);mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al.,Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and ahuman hepatoma line (Hep G2).

Host cells are transformed with the above-described expression orcloning vectors for antibody production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.

(viii) Culturing the Host Cells

The host cells used to produce an antibody of this invention may becultured in a variety of media. Commercially available media such asHam's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) aresuitable for culturing the host cells. In addition, any of the mediadescribed in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal.Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762;4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. Re.30,985 may be used as culture media for the host cells. Any of thesemedia may be supplemented as necessary with hormones and/or other growthfactors (such as insulin, transferrin, or epidermal growth factor),salts (such as sodium chloride, calcium, magnesium, and phosphate),buffers (such as HEPES), nucleotides (such as adenosine and thymidine),antibiotics (such as GENTAMYCIN™ drug), trace elements (defined asinorganic compounds usually present at final concentrations in themicromolar range), and glucose or an equivalent energy source. Any othernecessary supplements may also be included at appropriate concentrationsthat would be known to those skilled in the art. The culture conditions,such as temperature, pH, and the like, are those previously used withthe host cell selected for expression, and will be apparent to theordinarily skilled artisan.

(ix) Purification of Antibody

When using recombinant techniques, the antibody can be producedintracellularly, or directly secreted into the medium. If the antibodyis produced intracellularly, as a first step, the particulate debris,either host cells or lysed fragments, are removed, for example, bycentrifugation or ultrafiltration. Where the antibody is secreted intothe medium, supernatants from such expression systems are generallyfirst concentrated using a commercially available protein concentrationfilter, for example, an Amicon or Millipore Pellicon ultrafiltrationunit. A protease inhibitor such as PMSF may be included in any of theforegoing steps to inhibit proteolysis and antibiotics may be includedto prevent the growth of adventitious contaminants.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, gel electrophoresis,dialysis, and affinity chromatography, with affinity chromatographybeing the preferred purification technique. The suitability of protein Aas an affinity ligand depends on the species and isotype of anyimmunoglobulin Fc domain that is present in the antibody. Protein A canbe used to purify antibodies that are based on human γ1, γ2, or γ4 heavychains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G isrecommended for all mouse isotypes and for human γ3 (Guss et al., EMBOJ. 5:15671575 (1986)). The matrix to which the affinity ligand isattached is most often agarose, but other matrices are available.Mechanically stable matrices such as controlled pore glass orpoly(styrenedivinyl)benzene allow for faster flow rates and shorterprocessing times than can be achieved with agarose. Where the antibodycomprises a CH3 domain, the Bakerbond ABX™resin (J. T. Baker,Phillipsburg, N.J.) is useful for purification. Other techniques forprotein purification such as fractionation on an ion-exchange column,ethanol precipitation, Reverse Phase HPLC, chromatography on silica,chromatography on heparin SEPHAROSE™ chromatography on an anion orcation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

Following any preliminary purification step(s), the mixture comprisingthe antibody of interest and contaminants may be subjected to low pHhydrophobic interaction chromatography using an elution buffer at a pHbetween about 2.5-4.5, preferably performed at low salt concentrations(e.g., from about 0-0.25M salt).

Immunoconjugates

The invention also provides immunoconjugates (interchangeably termed“antibody-drug conjugates” or “ADC”), comprising any of the anti-Notch1NRR antibodies described herein conjugated to a cytotoxic agent such asa chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin(e.g., an enzymatically active toxin of bacterial, fungal, plant, oranimal origin, or fragments thereof), or a radioactive isotope (i.e., aradioconjugate).

The use of antibody-drug conjugates for the local delivery of cytotoxicor cytostatic agents, i.e., drugs to kill or inhibit tumor cells in thetreatment of cancer (Syrigos and Epenetos (1999) Anticancer Research19:605-614; Niculescu-Duvaz and Springer (1997) Adv. Drg. Del. Rev.26:151-172; U.S. Pat. No. 4,975,278) allows targeted delivery of thedrug moiety to tumors, and intracellular accumulation therein, wheresystemic administration of these unconjugated drug agents may result inunacceptable levels of toxicity to normal cells as well as the tumorcells sought to be eliminated (Baldwin et al., (1986) Lancet pp. (Mar.15, 1986):603-05; Thorpe, (1985) “Antibody Carriers Of Cytotoxic AgentsIn Cancer Therapy: A Review,” in Monoclonal Antibodies '84: BiologicalAnd Clinical Applications, A. Pinchera et al. (ed.s), pp. 475-506).Maximal efficacy with minimal toxicity is sought thereby. Bothpolyclonal antibodies and monoclonal antibodies have been reported asuseful in these strategies (Rowland et al., (1986) Cancer Immunol.Immunother., 21:183-87). Drugs used in these methods include daunomycin,doxorubicin, methotrexate, and vindesine (Rowland et al., (1986) supra).Toxins used in antibody-toxin conjugates include bacterial toxins suchas diphtheria toxin, plant toxins such as ricin, small molecule toxinssuch as geldanamycin (Mandler et al (2000) Jour. of the Nat. CancerInst. 92(19):1573-1581; Mandler et al., (2000) Bioorganic & Med. Chem.Letters 10:1025-1028; Mandler et al., (2002) Bioconjugate Chem.13:786-791), maytansinoids (EP 1391213; Liu et al., (1996) Proc. Natl.Acad. Sci. USA 93:8618-8623), and calicheamicin (Lode et al., (1998)Cancer Res. 58:2928; Hinman et al., (1993) Cancer Res. 53:3336-3342).The toxins may effect their cytotoxic and cytostatic effects bymechanisms including tubulin binding, DNA binding, or topoisomeraseinhibition. Some cytotoxic drugs tend to be inactive or less active whenconjugated to large antibodies or protein receptor ligands.

ZEVALIN® (ibritumomab tiuxetan, Biogen/Idec) is an antibody-radioisotopeconjugate composed of a murine IgG1 kappa monoclonal antibody directedagainst the CD20 antigen found on the surface of normal and malignant Blymphocytes and ¹¹¹In or ⁹⁰Y radioisotope bound by a thiourealinker-chelator (Wiseman et al., (2000) Eur. Jour. Nucl. Med.27(7):766-77; Wiseman et al., (2002) Blood 99(12):4336-42; Witzig etal., (2002) J. Clin. Oncol. 20(10):2453-63; Witzig et al., (2002) J.Clin. Oncol. 20(15):3262-69). Although ZEVALIN has activity againstB-cell non-Hodgkin's Lymphoma (NHL), administration results in severeand prolonged cytopenias in most patients. MYLOTARG™ (gemtuzumabozogamicin, Wyeth Pharmaceuticals), an antibody drug conjugate composedof a hu CD33 antibody linked to calicheamicin, was approved in 2000 forthe treatment of acute myeloid leukemia by injection (Drugs of theFuture (2000) 25(7):686; U.S. Pat. Nos. 4,970,198; 5,079,233; 5,585,089;5,606,040; 5,6937,62; 5,739,116; 5,767,285; 5,773,001). Cantuzumabmertansine (Immunogen, Inc.), an antibody drug conjugate composed of thehuC242 antibody linked via the disulfide linker SPP to the maytansinoiddrug moiety, DM1, is advancing into Phase II trials for the treatment ofcancers that express CanAg, such as colon, pancreatic, gastric, andothers. MLN-2704 (Millennium Pharm., BZL Biologics, Immunogen Inc.), anantibody drug conjugate composed of the anti-prostate specific membraneantigen (PSMA) monoclonal antibody linked to the maytansinoid drugmoiety, DM1, is under development for the potential treatment ofprostate tumors. The auristatin peptides, auristatin E (AE) andmonomethylauristatin (MMAE), synthetic analogs of dolastatin, wereconjugated to chimeric monoclonal antibodies cBR96 (specific to Lewis Yon carcinomas) and cAC10 (specific to CD30 on hematologicalmalignancies) (Doronina et al., (2003) Nature Biotechnology21(7):778-784) and are under therapeutic development.

Chemotherapeutic agents useful in the generation of immunoconjugates aredescribed herein (e.g., above). Enzymatically active toxins andfragments thereof that can be used include diphtheria A chain,nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.See, e.g., WO 93/21232 published Oct. 28, 1993. A variety ofradionuclides are available for the production of radioconjugatedantibodies. Examples include ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y, and ¹⁸⁶Re.Conjugates of the antibody and cytotoxic agent are made using a varietyof bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCl), active esters (such as disuccinimidyl suberate),aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science, 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026.

Conjugates of an antibody and one or more small molecule toxins, such asa calicheamicin, maytansinoids, dolastatins, aurostatins, atrichothecene, and CC1065, and the derivatives of these toxins that havetoxin activity, are also contemplated herein.

i. Maytansine and Maytansinoids

In some embodiments, the immunoconjugate comprises an antibody (fulllength or fragments) of the invention conjugated to one or moremaytansinoid molecules.

Maytansinoids are mitototic inhibitors which act by inhibiting tubulinpolymerization. Maytansine was first isolated from the east Africanshrub Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, it wasdiscovered that certain microbes also produce maytansinoids, such asmaytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042).Synthetic maytansinol and derivatives and analogues thereof aredisclosed, for example, in U.S. Pat. Nos. 4,137,230; 4,248,870;4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268;4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348;4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and4,371,533.

Maytansinoid drug moieties are attractive drug moieties in antibody drugconjugates because they are: (i) relatively accessible to prepare byfermentation or chemical modification, derivatization of fermentationproducts, (ii) amenable to derivatization with functional groupssuitable for conjugation through the non-disulfide linkers toantibodies, (iii) stable in plasma, and (iv) effective against a varietyof tumor cell lines.

Immunoconjugates containing maytansinoids, methods of making same, andtheir therapeutic use are disclosed, for example, in U.S. Pat. Nos.5,208,020, 5,416,064, and European Patent EP 0 425 235 B1, thedisclosures of which are hereby expressly incorporated by reference. Liuet al., Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996) describedimmunoconjugates comprising a maytansinoid designated DM1 linked to themonoclonal antibody C242 directed against human colorectal cancer. Theconjugate was found to be highly cytotoxic towards cultured colon cancercells, and showed antitumor activity in an in vivo tumor growth assay.Chari et al., Cancer Research 52:127-131 (1992) describeimmunoconjugates in which a maytansinoid was conjugated via a disulfidelinker to the murine antibody A7 binding to an antigen on human coloncancer cell lines, or to another murine monoclonal antibody TA.1 thatbinds the HER-2/neu oncogene. The cytotoxicity of the TA.1-maytansinoidconjugate was tested in vitro on the human breast cancer cell lineSK-BR-3, which expresses 3×10⁵ HER-2 surface antigens per cell. The drugconjugate achieved a degree of cytotoxicity similar to the freemaytansinoid drug, which could be increased by increasing the number ofmaytansinoid molecules per antibody molecule. The A7-maytansinoidconjugate showed low systemic cytotoxicity in mice.

Antibody-maytansinoid conjugates are prepared by chemically linking anantibody to a maytansinoid molecule without significantly diminishingthe biological activity of either the antibody or the maytansinoidmolecule. See, e.g., U.S. Pat. No. 5,208,020 (the disclosure of which ishereby expressly incorporated by reference). An average of 3-4maytansinoid molecules conjugated per antibody molecule has shownefficacy in enhancing cytotoxicity of target cells without negativelyaffecting the function or solubility of the antibody, although even onemolecule of toxin/antibody would be expected to enhance cytotoxicityover the use of naked antibody. Maytansinoids are well known in the artand can be synthesized by known techniques or isolated from naturalsources. Suitable maytansinoids are disclosed, for example, in U.S. Pat.No. 5,208,020 and in the other patents and nonpatent publicationsreferred to hereinabove. Preferred maytansinoids are maytansinol andmaytansinol analogues modified in the aromatic ring or at otherpositions of the maytansinol molecule, such as various maytansinolesters.

There are many linking groups known in the art for makingantibody-maytansinoid conjugates, including, for example, thosedisclosed in U.S. Pat. No. 5,208,020 or EP Patent 0 425 235 B1, Chari etal., Cancer Research 52:127-131 (1992), and U.S. patent application Ser.No. 10/960,602, filed Oct. 8, 2004, the disclosures of which are herebyexpressly incorporated by reference. Antibody-maytansinoid conjugatescomprising the linker component SMCC may be prepared as disclosed inU.S. patent application Ser. No. 10/960,602, filed Oct. 8, 2004. Thelinking groups include disulfide groups, thioether groups, acid labilegroups, photolabile groups, peptidase labile groups, or esterase labilegroups, as disclosed in the above-identified patents, disulfide andthioether groups being preferred. Additional linking groups aredescribed and exemplified herein.

Conjugates of the antibody and maytansinoid may be made using a varietyof bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). Particularly preferred coupling agentsinclude N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) (Carlssonet al., Biochem. J. 173:723-737 (1978)) andN-succinimidyl-4-(2-pyridylthio)pentanoate (SPP) to provide for adisulfide linkage.

The linker may be attached to the maytansinoid molecule at variouspositions, depending on the type of the link. For example, an esterlinkage may be formed by reaction with a hydroxyl group usingconventional coupling techniques. The reaction may occur at the C-3position having a hydroxyl group, the C-14 position modified withhydroxymethyl, the C-15 position modified with a hydroxyl group, and theC-20 position having a hydroxyl group. In a preferred embodiment, thelinkage is formed at the C-3 position of maytansinol or a maytansinolanalogue.

ii. Auristatins and Dolastatins

In some embodiments, the immunoconjugate comprises an antibody of theinvention conjugated to dolastatins or dolostatin peptidic analogs andderivatives, the auristatins (U.S. Pat. Nos. 5,635,483 and 5,780,588).Dolastatins and auristatins have been shown to interfere withmicrotubule dynamics, GTP hydrolysis, and nuclear and cellular division(Woyke et al (2001) Antimicrob. Agents and Chemother. 45(12):3580-3584)and have anticancer (U.S. Pat. No. 5,663,149) and antifungal activity(Pettit et al., (1998) Antimicrob. Agents Chemother. 42:2961-2965). Thedolastatin or auristatin drug moiety may be attached to the antibodythrough the N (amino) terminus or the C (carboxyl) terminus of thepeptidic drug moiety (WO 02/088172).

Exemplary auristatin embodiments include the N-terminus linkedmonomethylauristatin drug moieties DE and DF, disclosed in“Monomethylvaline Compounds Capable of Conjugation to Ligands,” U.S.Ser. No. 10/983,340, filed Nov. 5, 2004, the disclosure of which isexpressly incorporated by reference in its entirety.

Typically, peptide-based drug moieties can be prepared by forming apeptide bond between two or more amino acids and/or peptide fragments.Such peptide bonds can be prepared, for example, according to the liquidphase synthesis method (see E. Schröder and K. Lübke, “The Peptides,”volume 1, pp. 76-136, 1965, Academic Press) that is well known in thefield of peptide chemistry. The auristatin/dolastatin drug moieties maybe prepared according to the methods of: U.S. Pat. Nos. 5,635,483 and5,780,588; Pettit et al., (1989) J. Am. Chem. Soc. 111:5463-5465; Pettitet al., (1998) Anti-Cancer Drug Design 13:243-277; Pettit, G. R., etal., Synthesis, 1996, 719-725; and Pettit et al., (1996) J. Chem. Soc.Perkin Trans. 1 5:859-863. See also Doronina (2003) Nat. Biotechnol.21(7):778-784; “Monomethylvaline Compounds Capable of Conjugation toLigands,” U520050238649, published Oct. 27, 2005, hereby incorporated byreference in its entirety (disclosing, e.g., linkers and methods ofpreparing monomethylvaline compounds such as MMAE and MMAF conjugated tolinkers).

iii. Calicheamicin

In other embodiments, the immunoconjugate comprises an antibody of theinvention conjugated to one or more calicheamicin molecules. Thecalicheamicin family of antibiotics are capable of producingdouble-stranded DNA breaks at sub-picomolar concentrations. For thepreparation of conjugates of the calicheamicin family, see U.S. Pat.Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710,5,773,001, and 5,877,296 (all to American Cyanamid Company). Structuralanalogues of calicheamicin which may be used include, but are notlimited to, γ₁ ^(I), α₂ ^(I), α₃ ^(I), N-acetyl-γ₁ ^(I), PSAG and θ^(I)₁ (Hinman et al., Cancer Research 53:3336-3342 (1993), Lode et al.,Cancer Research 58:2925-2928 (1998) and the aforementioned U.S. patentsto American Cyanamid). Another anti-tumor drug that the antibody can beconjugated is QFA which is an antifolate. Both calicheamicin and QFAhave intracellular sites of action and do not readily cross the plasmamembrane. Therefore, cellular uptake of these agents through antibodymediated internalization greatly enhances their cytotoxic effects.

iv. Other Cytotoxic Agents

Other antitumor agents that can be conjugated to the antibodies of theinvention include BCNU, streptozoicin, vincristine and 5-fluorouracil,the family of agents known collectively LL-E33288 complex described inU.S. Pat. Nos. 5,053,394 and 5,770,710, as well as esperamicins (U.S.Pat. No. 5,877,296).

Enzymatically active toxins and fragments thereof which can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), momordica charantia inhibitor, curcin, crotin, Sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin and the tricothecenes. See, for example, WO 93/21232 publishedOct. 28, 1993.

The present invention further contemplates an immunoconjugate formedbetween an antibody and a compound with nucleolytic activity (e.g., aribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase).

For selective destruction of the tumor, the antibody may comprise ahighly radioactive atom. A variety of radioactive isotopes are availablefor the production of radioconjugated antibodies. Examples includeAt²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² andradioactive isotopes of Lu. When the conjugate is used for detection, itmay comprise a radioactive atom for scintigraphic studies, for exampletc^(99m) or I¹²³, or a spin label for nuclear magnetic resonance (NMR)imaging (also known as magnetic resonance imaging, mri), such asiodine-123 again, iodine-131, indium-111, fluorine-19, carbon-13,nitrogen-15, oxygen-17, gadolinium, manganese or iron.

The radio- or other labels may be incorporated in the conjugate in knownways. For example, the peptide may be biosynthesized or may besynthesized by chemical amino acid synthesis using suitable amino acidprecursors involving, for example, fluorine-19 in place of hydrogen.Labels such as Tc^(99m) or I¹²³, Re¹⁸⁶, Re¹⁸⁸ and In¹¹¹ can be attachedvia a cysteine residue in the peptide. Yttrium-90 can be attached via alysine residue. The IODOGEN method (Fraker et al (1978) Biochem.Biophys. Res. Commun. 80: 49-57 can be used to incorporate iodine-123.“Monoclonal Antibodies in Immunoscintigraphy” (Chatal, CRC Press 1989)describes other methods in detail.

Conjugates of the antibody and cytotoxic agent may be made using avariety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238:1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026. Thelinker may be a “cleavable linker” facilitating release of the cytotoxicdrug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, photolabile linker, dimethyl linker ordisulfide-containing linker (Chari et al., Cancer Research 52:127-131(1992); U.S. Pat. No. 5,208,020) may be used.

The compounds of the invention expressly contemplate, but are notlimited to, ADC prepared with cross-linker reagents: BMPS, EMCS, GMBS,HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS,sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, andsulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate) which arecommercially available (e.g., from Pierce Biotechnology, Inc., Rockford,Ill., U.S.A). See pages 467-498, 2003-2004 Applications Handbook andCatalog.

v. Preparation of Antibody Drug Conjugates

In the antibody drug conjugates (ADC) of the invention, an antibody (Ab)is conjugated to one or more drug moieties (D), e.g. about 1 to about 20drug moieties per antibody, through a linker (L). The ADC of Formula Imay be prepared by several routes, employing organic chemistryreactions, conditions, and reagents known to those skilled in the art,including: (1) reaction of a nucleophilic group of an antibody with abivalent linker reagent, to form Ab-L, via a covalent bond, followed byreaction with a drug moiety D; and (2) reaction of a nucleophilic groupof a drug moiety with a bivalent linker reagent, to form D-L, via acovalent bond, followed by reaction with the nucleophilic group of anantibody. Additional methods for preparing ADC are described herein.

Ab-(L-D)_(P)

The linker may be composed of one or more linker components. Exemplarylinker components include 6-maleimidocaproyl (“MC”), maleimidopropanoyl(“MP”), valine-citrulline (“val-cit”), alanine-phenylalanine(“ala-phe”), p-aminobenzyloxycarbonyl (“PAB”), N-Succinimidyl4-(2-pyridylthio)pentanoate (“SPP”), N-Succinimidyl4-(N-maleimidomethyl)cyclohexane-1 carboxylate (“SMCC”), andN-Succinimidyl (4-iodo-acetyl)aminobenzoate (“SIAB”). Additional linkercomponents are known in the art and some are described herein. See also“Monomethylvaline Compounds Capable of Conjugation to Ligands,” U.S.Ser. No. 10/983,340, filed Nov. 5, 2004, the contents of which arehereby incorporated by reference in its entirety.

In some embodiments, the linker may comprise amino acid residues.Exemplary amino acid linker components include a dipeptide, atripeptide, a tetrapeptide or a pentapeptide. Exemplary dipeptidesinclude: valine-citrulline (vc or val-cit), alanine-phenylalanine (af orala-phe). Exemplary tripeptides include: glycine-valine-citrulline(gly-val-cit) and glycine-glycine-glycine (gly-gly-gly) Amino acidresidues which comprise an amino acid linker component include thoseoccurring naturally, as well as minor amino acids and non-naturallyoccurring amino acid analogs, such as citrulline Amino acid linkercomponents can be designed and optimized in their selectivity forenzymatic cleavage by a particular enzymes, for example, atumor-associated protease, cathepsin B, C and D, or a plasmin protease.

Nucleophilic groups on antibodies include, but are not limited to: (i)N-terminal amine groups, (ii) side chain amine groups, e.g., lysine,(iii) side chain thiol groups, e.g., cysteine, and (iv) sugar hydroxylor amino groups where the antibody is glycosylated. Amine, thiol, andhydroxyl groups are nucleophilic and capable of reacting to formcovalent bonds with electrophilic groups on linker moieties and linkerreagents including: (i) active esters such as NHS esters, HOBt esters,haloformates, and acid halides; (ii) alkyl and benzyl halides such ashaloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimidegroups. Certain antibodies have reducible interchain disulfides, i.e.,cysteine bridges. Antibodies may be made reactive for conjugation withlinker reagents by treatment with a reducing agent such as DTT(dithiothreitol). Each cysteine bridge will thus form, theoretically,two reactive thiol nucleophiles. Additional nucleophilic groups can beintroduced into antibodies through the reaction of lysines with2-iminothiolane (Traut's reagent) resulting in conversion of an amineinto a thiol. Reactive thiol groups may be introduced into the antibody(or fragment thereof) by introducing one, two, three, four, or morecysteine residues (e.g., preparing mutant antibodies comprising one ormore non-native cysteine amino acid residues).

Antibody drug conjugates of the invention may also be produced bymodification of the antibody to introduce electrophilic moieties, whichcan react with nucleophilic substituents on the linker reagent or drug.The sugars of glycosylated antibodies may be oxidized, e.g., withperiodate oxidizing reagents, to form aldehyde or ketone groups whichmay react with the amine group of linker reagents or drug moieties. Theresulting imine Schiff base groups may form a stable linkage, or may bereduced, e.g., by borohydride reagents to form stable amine linkages. Inone embodiment, reaction of the carbohydrate portion of a glycosylatedantibody with either glactose oxidase or sodium meta-periodate may yieldcarbonyl (aldehyde and ketone) groups in the protein that can react withappropriate groups on the drug (Hermanson, Bioconjugate Techniques). Inanother embodiment, proteins containing N-terminal serine or threonineresidues can react with sodium meta-periodate, resulting in productionof an aldehyde in place of the first amino acid (Geoghegan & Stroh,(1992) Bioconjugate Chem. 3:138-146; U.S. Pat. No. 5,362,852). Suchaldehyde can be reacted with a drug moiety or linker nucleophile.

Likewise, nucleophilic groups on a drug moiety include, but are notlimited to: amine, thiol, hydroxyl, hydrazide, oxime, hydrazine,thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groupscapable of reacting to form covalent bonds with electrophilic groups onlinker moieties and linker reagents including: (i) active esters such asNHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl andbenzyl halides such as haloacetamides; (iii) aldehydes, ketones,carboxyl, and maleimide groups.

Alternatively, a fusion protein comprising the antibody and cytotoxicagent may be made, e.g., by recombinant techniques or peptide synthesis.The length of DNA may comprise respective regions encoding the twoportions of the conjugate either adjacent one another or separated by aregion encoding a linker peptide which does not destroy the desiredproperties of the conjugate.

In yet another embodiment, the antibody may be conjugated to a“receptor” (such streptavidin) for utilization in tumor pre-targetingwherein the antibody-receptor conjugate is administered to theindividual, followed by removal of unbound conjugate from thecirculation using a clearing agent and then administration of a “ligand”(e.g., avidin) which is conjugated to a cytotoxic agent (e.g., aradionucleotide).

Pharmaceutical Formulations

Therapeutic formulations comprising an antibody of the invention areprepared for storage by mixing the antibody having the desired degree ofpurity with optional physiologically acceptable carriers, excipients orstabilizers (Remington: The Science and Practice of Pharmacy 20thedition (2000)), in the form of aqueous solutions, lyophilized or otherdried formulations. Acceptable carriers, excipients, or stabilizers arenontoxic to recipients at the dosages and concentrations employed, andinclude buffers such as phosphate, citrate, histidine and other organicacids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride, benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g., Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.Such molecules are suitably present in combination in amounts that areeffective for the purpose intended.

The active ingredients may also be entrapped in microcapsule prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington: The Science and Practice of Pharmacy 20th edition (2000).

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semi-permeable matrices of solidhydrophobic polymers containing the immunoglobulin of the invention,which matrices are in the form of shaped articles, e.g., films, ormicrocapsule. Examples of sustained-release matrices include polyesters,hydrogels (for example, poly(2-hydroxyethyl-methacrylate), orpoly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymersof L-glutamic acid and γ ethyl-L-glutamate, non-degradableethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymerssuch as the LUPRON DEPOT™ (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), andpoly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinylacetate and lactic acid-glycolic acid enable release of molecules forover 100 days, certain hydrogels release proteins for shorter timeperiods. When encapsulated immunoglobulins remain in the body for a longtime, they may denature or aggregate as a result of exposure to moistureat 37° C., resulting in a loss of biological activity and possiblechanges in immunogenicity. Rational strategies can be devised forstabilization depending on the mechanism involved. For example, if theaggregation mechanism is discovered to be intermolecular S—S bondformation through thio-disulfide interchange, stabilization may beachieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

III. Therapeutic Uses

The antibodies and antibody fragments described herein which bind bothHER2 and VEGF (e.g., bH1-44 or bH1-88 or fragments thereof) can be usedfor the treatment of tumors, including pre-cancerous, non-metastatic,and cancerous tumors (e.g., early stage cancer), for the treatment ofautoimmune disease, for the treatment of an angiogenesis disorder, forthe treatment of a disease involving abnormal activation of HER2, or forthe treatment of a subject at risk for developing cancer (for example,breast cancer, colorectal cancer, lung cancer, renal cell carcinoma,glioma, or ovarian cancer), an angiogenesis disorder, an autoimmunedisease, or a disease involving abnormal activation of HER2.

The term cancer embraces a collection of proliferative disorders,including but not limited to pre-cancerous growths, benign tumors, andmalignant tumors. Benign tumors remain localized at the site of originand do not have the capacity to infiltrate, invade, or metastasize todistant sites. Malignant tumors will invade and damage other tissuesaround them. They can also gain the ability to break off from where theystarted and spread to other parts of the body (metastasize), usuallythrough the bloodstream or through the lymphatic system where the lymphnodes are located. Primary tumors are classified by the type of tissuefrom which they arise; metastatic tumors are classified by the tissuetype from which the cancer cells are derived. Over time, the cells of amalignant tumor become more abnormal and appear less like normal cells.This change in the appearance of cancer cells is called the tumor gradeand cancer cells are described as being well-differentiated,moderately-differentiated, poorly-differentiated, or undifferentiated.Well-differentiated cells are quite normal appearing and resemble thenormal cells from which they originated. Undifferentiated cells arecells that have become so abnormal that it is no longer possible todetermine the origin of the cells.

The tumor can be a solid tumor or a non-solid or soft tissue tumor.Examples of soft tissue tumors include leukemia (e.g., chronicmyelogenous leukemia, acute myelogenous leukemia, adult acutelymphoblastic leukemia, acute myelogenous leukemia, mature B-cell acutelymphoblastic leukemia, chronic lymphocytic leukemia, polymphocyticleukemia, or hairy cell leukemia), or lymphoma (e.g., non-Hodgkin'slymphoma, cutaneous T-cell lymphoma, or Hodgkin's disease). A solidtumor includes any cancer of body tissues other than blood, bone marrow,or the lymphatic system. Solid tumors can be further separated intothose of epithelial cell origin and those of non-epithelial cell origin.Examples of epithelial cell solid tumors include tumors of thegastrointestinal tract, colon, breast, prostate, lung, kidney, liver,pancreas, ovary, head and neck, oral cavity, stomach, duodenum, smallintestine, large intestine, anus, gall bladder, labium, nasopharynx,skin, uterus, male genital organ, urinary organs, bladder, and skin.Solid tumors of non-epithelial origin include sarcomas, brain tumors,and bone tumors.

Epithelial cancers generally evolve from a benign tumor to a preinvasivestage (e.g., carcinoma in situ), to a malignant cancer, which haspenetrated the basement membrane and invaded the subepithelial stroma.

Multispecific antibodies that bind both VEGF and HER2 (e.g., bH1-44 orbH1-88 or a fragment thereof) desirably are used to treat breast cancer,colorectal cancer, lung cancer, renal cell carcinoma, glioma, or ovariancancer.

It is now well established that angiogenesis is implicated in thepathogenesis of a variety of disorders. These include solid tumors andmetastasis, atherosclerosis, retrolental fibroplasia, hemangiomas,chronic inflammation, intraocular neovascular diseases such asproliferative retinopathies, e.g., diabetic retinopathy, age-relatedmacular degeneration (AMD), neovascular glaucoma, immune rejection oftransplanted corneal tissue and other tissues, rheumatoid arthritis, andpsoriasis. Folkman et al., J. Biol. Chem., 267:10931-10934 (1992);Klagsbrun et al., Annu. Rev. Physiol. 53:217-239 (1991); and Garner A.,“Vascular diseases”, In: Pathobiology of Ocular Disease. A DynamicApproach, Garner A., Klintworth G K, eds., 2nd Edition (Marcel Dekker,NY, 1994), pp 1625-1710.

Abnormal angiogenesis occurs when new blood vessels either growexcessively, insufficiently or inappropriately (e.g., the location,timing or onset of the angiogenesis being undesired from a medicalstandpoint) in a diseased state or such that it causes a diseased state.Excessive, inappropriate or uncontrolled angiogenesis occurs when thereis new blood vessel growth that contributes to the worsening of thediseased state or causes a diseased state, such as in cancer, especiallyvascularized solid tumors and metastatic tumors (including colon, lungcancer (especially small-cell lung cancer), or prostate cancer),diseases caused by ocular neovascularization, especially diabeticblindness, retinopathies, primarily diabetic retinopathy or age-relatedmacular degeneration (AMD), diabetic macular edema, cerebral edema(e.g., associated with acute stroke/closed head injury/trauma), synovialinflammation, pannus formation in rheumatoid arthritis, myositisossificans, hypertropic bone formation, refractory ascites, polycysticovarian disease, 3rd spacing of fluid diseases (pancreatitis,compartment syndrome, burns, bowel disease), uterine fibroids, prematurelabor, neovascularization of the angle (rubeosis), malignant pulmonaryeffusions, vascular restenosis, haemangioblastoma such as haemangioma;inflammatory renal diseases, such as glomerulonephritis, especiallymesangioproliferative glomerulonephritis, haemolytic uremic syndrome,diabetic nephropathy or hypertensive nephrosclerosis, variousinflammatory diseases, such as arthritis, especially rheumatoidarthritis, inflammatory bowel disease, psoriasis, psoriatic arthritis,psoriatic plaques, sarcoidosis, arterial arteriosclerosis, and diseasesoccurring after transplants, renal allograft rejection, endometriosis orchronic asthma, and more than 70 other conditions. The new blood vesselscan feed the diseased tissues, destroy normal tissues, and in the caseof cancer, the new vessels can allow tumor cells to escape into thecirculation and lodge in other organs (tumor metastases). Insufficientangiogenesis occurs when there is inadequate blood vessels growth thatcontributes to the worsening of a diseased state, e.g., in diseases suchas coronary artery disease, stroke, and delayed wound healing. Further,ulcers, strokes, and heart attacks can result from the absence ofangiogenesis that normally is required for natural healing. The presentinvention contemplates treating those patients that have or are at riskof developing the above-mentioned illnesses using an antibody thatspecifically binds both VEGF and HER2 (e.g., the bH1-81 or bH1-44antibody).

Other patients that are candidates for receiving compositions of thisinvention have, or are at risk for developing, abnormal proliferation offibrovascular tissue, acne rosacea, acquired immune deficiency syndrome,artery occlusion, atopic keratitis, bacterial ulcers, Bechets disease,blood borne tumors, carotid obstructive disease, choroidalneovascularization, chronic inflammation, chronic retinal detachment,chronic uveitis, chronic vitritis, contact lens overwear, corneal graftrejection, corneal neovascularization, corneal graft neovascularization,Crohn's disease, Eales disease, epidemic keratoconjunctivitis, fungalulcers, Herpes simplex infections, Herpes zoster infections,hyperviscosity syndromes, Kaposi's sarcoma, leukemia, lipiddegeneration, Lyme's disease, marginal keratolysis, Mooren ulcer,Mycobacteria infections other than leprosy, myopia, ocular neovasculardisease, optic pits, Osler-Weber syndrome (Osler-Weber-Rendu),osteoarthritis, Paget's disease, pars planitis, pemphigoid,phylectenulosis, polyarteritis, post-laser complications, protozoaninfections, pseudoxanthoma elasticum, pterygium keratitis sicca, radialkeratotomy, retinal neovascularization, retinopathy of prematurity,retrolental fibroplasias, sarcoid, scleritis, sickle cell anemia,Sogren's syndrome, solid tumors, Stargart's disease, Steven's Johnsondisease, superior limbic keratitis, syphilis, systemic lupus, Terrien'smarginal degeneration, toxoplasmosis, tumors of Ewing sarcoma, tumors ofneuroblastoma, tumors of osteosarcoma, tumors of retinoblastoma, tumorsof rhabdomyosarcoma, ulcerative colitis, vein occlusion, Vitamin Adeficiency, Wegener's sarcoidosis, undesired angiogenesis associatedwith diabetes, parasitic diseases, abnormal wound healing, hypertrophyfollowing surgery, injury or trauma (e.g., acute lung injury/ARDS),inhibition of hair growth, inhibition of ovulation and corpus luteumformation, inhibition of implantation, and inhibition of embryodevelopment in the uterus.

Anti-angiogenesis therapies are useful in the general treatment of graftrejection, lung inflammation, primary pulmonary hypertension, nephroticsyndrome, preeclampsia, and pleural effusion, diseases and disorderscharacterized by undesirable vascular permeability, e.g., edemaassociated with brain tumors, ascites associated with malignancies,Meigs' syndrome, lung inflammation, nephrotic syndrome, pericardialeffusion (such as associated with pericarditis), permeability associatedwith cardiovascular diseases such as the condition following myocardialinfarctions and strokes and the like, and sepsis.

Other angiogenesis-dependent diseases according to this inventioninclude angiofibroma (abnormal blood of vessels which are prone tobleeding), neovascular glaucoma (growth of blood vessels in the eye),arteriovenous malformations (AVM; abnormal communication betweenarteries and veins), nonunion fractures (fractures that will not heal),atherosclerotic plaques (hardening of the arteries), pyogenic granuloma(common skin lesion composed of blood vessels), scleroderma (a form ofconnective tissue disease), hemangioma (tumor composed of bloodvessels), meningioma, thyroid hyperplasias (including Grave's disease),trachoma (leading cause of blindness in the third world), hemophilicjoints, synovitis, dermatitis, vascular adhesions, and hypertrophicscars (abnormal scar formation).

IV. Dosages and Formulations

The antibody (e.g., bH1-44 or bH1-81) or antibody fragment compositionswill be formulated, dosed, and administered in a fashion consistent withgood medical practice. Factors for consideration in this context includethe particular disorder being treated, the particular mammal beingtreated, the clinical condition of the individual subject, the cause ofthe disorder, the site of delivery of the agent, the method ofadministration, the scheduling of administration, and other factorsknown to medical practitioners. The “therapeutically effective amount”of the antibody or antibody fragment to be administered will be governedby such considerations, and is the minimum amount necessary to prevent,ameliorate, or treat a cancer or autoimmune disorder. The antibody orantibody fragment need not be, but is optionally, formulated with one ormore agents currently used to prevent or treat cancer or an autoimmunedisorder or a risk of developing cancer or an autoimmune disorder. Theeffective amount of such other agents depends on the amount of antibodyor antibody fragment present in the formulation, the type of disorder ortreatment, and other factors discussed above. These are generally usedin the same dosages and with administration routes as used hereinbeforeor about from 1 to 99% of the heretofore employed dosages. Generally,alleviation or treatment of a cancer involves the lessening of one ormore symptoms or medical problems associated with the cancer. Thetherapeutically effective amount of the drug can accomplish one or acombination of the following: reduce (by at least 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 100% or more) the number of cancer cells;reduce or inhibit the tumor size or tumor burden; inhibit (i.e., todecrease to some extent and/or stop) cancer cell infiltration intoperipheral organs; reduce hormonal secretion in the case of adenomas;reduce vessel density; inhibit tumor metastasis; reduce or inhibit tumorgrowth; and/or relieve to some extent one or more of the symptomsassociated with the cancer. In some embodiments, the antibody orantibody fragment is used to prevent the occurrence or reoccurrence ofcancer or an autoimmune disorder in the subject.

In one embodiment, the present invention can be used for increasing theduration of survival of a human patient susceptible to or diagnosed witha cancer or autoimmune disorder. Duration of survival is defined as thetime from first administration of the drug to death. Duration ofsurvival can also be measured by stratified hazard ratio (HR) of thetreatment group versus control group, which represents the risk of deathfor a patient during the treatment.

In yet another embodiment, the treatment of the present inventionsignificantly increases response rate in a group of human patientssusceptible to or diagnosed with a cancer who are treated with variousanti-cancer therapies. Response rate is defined as the percentage oftreated patients who responded to the treatment. In one aspect, thecombination treatment of the invention using an antibody or antibodyfragment and surgery, radiation therapy, or one or more chemotherapeuticagents significantly increases response rate in the treated patientgroup compared to the group treated with surgery, radiation therapy, orchemotherapy alone, the increase having a Chi-square p-value of lessthan 0.005.

Additional measurements of therapeutic efficacy in the treatment ofcancers are described in U.S. Patent Application Publication No.20050186208.

Therapeutic formulations are prepared using standard methods known inthe art by mixing the active ingredient having the desired degree ofpurity with optional physiologically acceptable carriers, excipients orstabilizers (Remington's Pharmaceutical Sciences (20^(th) edition), ed.A. Gennaro, 2000, Lippincott, Williams & Wilkins, Philadelphia, Pa.).Acceptable carriers, include saline, or buffers such as phosphate,citrate and other organic acids; antioxidants including ascorbic acid;low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilicpolymers such as polyvinylpyrrolidone, amino acids such as glycine,glutamine, asparagines, arginine or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugar alcohols such as mannitolor sorbitol; salt-forming counterions such as sodium; and/or nonionicsurfactants such as TWEEN™, PLURONICS™, or PEG.

Optionally, but preferably, the formulation contains a pharmaceuticallyacceptable salt, preferably sodium chloride, and preferably at aboutphysiological concentrations. Optionally, the formulations of theinvention can contain a pharmaceutically acceptable preservative. Insome embodiments the preservative concentration ranges from 0.1 to 2.0%,typically v/v. Suitable preservatives include those known in thepharmaceutical arts. Benzyl alcohol, phenol, m-cresol, methylparaben,and propylparaben are preferred preservatives. Optionally, theformulations of the invention can include a pharmaceutically acceptablesurfactant at a concentration of 0.005 to 0.02%.

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.Such molecules are suitably present in combination in amounts that areeffective for the purpose intended.

The active ingredients may also be entrapped in microcapsule prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences, supra.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, or microcapsule. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods. When encapsulated antibodies remainin the body for a long time, they may denature or aggregate as a resultof exposure to moisture at 37° C., resulting in a loss of biologicalactivity and possible changes in immunogenicity. Rational strategies canbe devised for stabilization depending on the mechanism involved. Forexample, if the aggregation mechanism is discovered to be intermolecularS—S bond formation through thio-disulfide interchange, stabilization maybe achieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

The antibodies and antibody fragments described herein (e.g., bH1-44 orbH1-81 or fragments thereof) are administered to a human subject, inaccord with known methods, such as intravenous administration as a bolusor by continuous infusion over a period of time, by intramuscular,intraperitoneal, intracerobrospinal, subcutaneous, intra-articular,intrasynovial, intrathecal, oral, topical, or inhalation routes. Localadministration may be particularly desired if extensive side effects ortoxicity is associated with VEGF and/or HER2 antagonism. An ex vivostrategy can also be used for therapeutic applications. Ex vivostrategies involve transfecting or transducing cells obtained from thesubject with a polynucleotide encoding an antibody or antibody fragment.The transfected or transduced cells are then returned to the subject.The cells can be any of a wide range of types including, withoutlimitation, hemopoietic cells (e.g., bone marrow cells, macrophages,monocytes, dendritic cells, T cells, or B cells), fibroblasts,epithelial cells, endothelial cells, keratinocytes, or muscle cells.

In one example, the antibody (e.g., bH1-44 or bH1-81) or antibodyfragment is administered locally, e.g., by direct injections, when thedisorder or location of the tumor permits, and the injections can berepeated periodically. The antibody or antibody fragment can also bedelivered systemically to the subject or directly to the tumor cells,e.g., to a tumor or a tumor bed following surgical excision of thetumor, in order to prevent or reduce local recurrence or metastasis.

V. Articles of Manufacture and Kits

Another embodiment of the invention is an article of manufacturecontaining materials useful for the treatment of autoimmune diseases andcancers. The article of manufacture comprises a container and a label orpackage insert on or associated with the container. Suitable containersinclude, for example, bottles, vials, syringes, etc. The containers maybe formed from a variety of materials such as glass or plastic. Thecontainer holds a composition which is effective for treating thecondition and may have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). At least one active agent in thecomposition is a multispecific antibody or antibody fragment antibody ofthe invention. The label or package insert indicates that thecomposition is used for treating the particular condition. The label orpackage insert will further comprise instructions for administering theantibody composition to the patient. Articles of manufacture and kitscomprising combinatorial therapies described herein are alsocontemplated.

Package insert refers to instructions customarily included in commercialpackages of therapeutic products that contain information about theindications, usage, dosage, administration, contraindications and/orwarnings concerning the use of such therapeutic products. In otherembodiments, the package insert indicates that the composition is usedfor treating breast cancer, colorectal cancer, lung cancer, renal cellcarcinoma, glioma, or ovarian cancer.

Additionally, the article of manufacture may further comprise a secondcontainer comprising a pharmaceutically-acceptable buffer, such asbacteriostatic water for injection (BWH), phosphate-buffered saline,Ringer's solution and dextrose solution. It may further include othermaterials from a commercial and user standpoint, including otherbuffers, diluents, filters, needles, and syringes.

Kits are also provided that are useful for various purposes, e.g., forpurification or immunoprecipitation of VEGF or HER2 from cells. Forisolation and purification of VEGF, or HER2, the kit can contain aVEGF/HER2 antibody (e.g., bH1-44 or bH1-81) coupled to beads (e.g.,sepharose beads). Kits can be provided which contain the antibodies fordetection and quantitation of VEGF or HER2 in vitro, e.g., in an ELISAor a Western blot. As with the article of manufacture, the kit comprisesa container and a label or package insert on or associated with thecontainer. The container holds a composition comprising at least onemultispecific antibody or antibody fragment of the invention. Additionalcontainers may be included that contain, e.g., diluents and buffers orcontrol antibodies. The label or package insert may provide adescription of the composition as well as instructions for the intendedin vitro or diagnostic use.

The foregoing written description is considered to be sufficient toenable one skilled in the art to practice the invention. The followingExamples are offered for illustrative purposes only, and are notintended to limit the scope of the present invention in any way. Indeed,various modifications of the invention in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description and fall within the scope of the appendedclaims.

Examples Example 1 Library Design and Construction

The antigen-binding site of antibody is formed by the association of thevariable domain (V_(H), V_(L)) of heavy chain (HC) and light chain (LC),each containing three CDR loops for antigen recognition. In many casesone of the two variable domains, often V_(H), determines the antigenspecificity. Mice with transgenic HC but intact LC repertoire generateneutralizing antibody titers (Senn et al., Eur. J. Immunol. 33:950-961,2003). We set out to investigate how bi-specificity of an antibody canoccur and whether different utilization of the V_(H) and the V_(L)domains can enable dual antigen binding specificity.

A semi-empirical approach was taken to find a design for diversifyingthe amino acid composition and CDR length of antibody light chain and alibrary template that enabled generation of a functional phage-displayedantibody library from which antibodies binding specifically to a proteinantigen could be selected. The sequence and length diversity of the CDRregions of approximately 1500 human kappa light chain sequences, asrepresented in the Kabat database, served to guide the library designprocess. Solvent exposed residues were targeted for randomization. Asubset of the randomized positions were tailored to represent aminoacids which are part of the natural repertoire at these sites, whereasthe remaining sites were randomized to include all 20 naturallyoccurring amino acids.

In particular, the light chain template (variable domain) set forthbelow was modified as described herein (underlined residues arerandomized) (SEQ ID NO:10).

DIQMTQSPSSLSASVGDRVTITCRASQD²⁸VNTAVAWYQQKPGKAPKWYS⁵⁰ASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQH⁹¹YTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGECFour sets of libraries were generated based on 3 human Fab and scFvtemplates where distinct sets of positions were targeted forrandomization (FIG. 1).

In all of the libraries the heavy chain was held constant with itssequence defined by the library template. The heavy chain template(variable domain) sequence is set forth below (SEQ ID NO:11).

EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH

The library designs are summarized in FIG. 1 and FIG. 2. All librarytemplates contained a stop codon (Sidhu et al., 2004) embedded in CDR L1preventing the presence of template light chain among thephage-displayed antibody library members. The template CDR sequences aresummarized in FIG. 3.

In one example, we introduced mutations in the LC variable domain of aHER2-specific antibody to identify variants that can bind a differentprotein antigen while retaining the original binding specificity. Wetook a conservative approach to randomize the LC CDRs in order togenerate variants that can be stably expressed. Twelve solvent exposedLC CDR positions were selected for randomization: five in CDR1 (28, 29,30, 31, 32), three in CDR2 (50, 51, 53) and four in CDR3 (91, 92, 93,94). Further, to guide the design of amino acid diversity at electedsites, the natural diversity of these positions was examined by analysisof approximately 1500 human kappa LC CDR sequences (Johnson and Wu,Nucleic Acids Res. 28:214, 2000; Chothia and Lesk, J. Mol. Biol.196:901, 1987) (FIG. 4). Some positions with relatively high naturaldiversity (30, 31, 50, 92, 93) were fully randomized while otherpositions were limited to as few as two amino acid types to mimicnatural antibodies. The length variation of natural LC CDR1 and CDR3 wasalso reflected in the library (FIG. 4). In FIG. 4, X denotes the aminoacid types designed at low frequencies as shown. Length diversity isconstructed by inserting 1 to 5 residues between residues 30 and 31 andbetween residues 93 and 94.

The LC library is a productive naive repertoire (Table 1). Listed areresults from the screening of 95 random clones at the end of four roundsof selection. In particular, selection for new binding specificity wasperformed as described on immobilized targets (VEGF, DR5, and human Fc)(Sidhu et al., J. Mol. Biol. 338:299, 2004). After four rounds ofselection 95 phage clones were assayed using ELISA for binding to thetarget, HER2, and a non-target protein, BSA, to ensure specific binding.To enrich for target binding clones that maintained HER2 binding, afinal round of selection on HER2 was performed. The positive clones weresequenced. To identify the highest affinity binders, the IC₅₀ forantigen binding was determined by competitive ELISA (Sidhu et al., J.Mol. Biol. 338:299, 2004). The number of unique clones as determined bysequence analysis and the number of unique clones that maintain HER2binding (bispecific clones) are shown. These clones show minimumbackground binding signals to irrelevant antigens, such as BSA.

TABLE 1 Light chain library selection summary Positive % Unique Seq.HER2 positive Human Fc fusion 63 31 out of 61 1 hVEGF 77 41 out of 74 30out of 41 DR5 long 85  5 out of 82 2* out of 5  *= weak binding signalTarget Bi-Specific, Screen Bi-Specific, Selection Human Fc fusion  1 outof 31 Not determined hVEGF 30 out of 41 94 out of 94 DR5 long 2* out of5   2 out of 7** *= weak binding signal Her2 **= weak binding signal DR5

Selection against three protein antigens: human vascular endothelialgrowth factor (hVEGF), death receptor 5 (DR5), and complement bindingfragment of IgG (Fc) generated many binding clones (FIG. 5A). Someclones lost binding affinity for HER2, while others maintainedHER2-binding and were thus bi-specific. Sequence analysis of the 131unique Herceptin® antibody variants with new binding specificityidentified the amino acid substitutions and insertions compared to theHerceptin® antibody (FIG. 5B).

The number of mutations ranged from 3-17. The clones that retained HER2binding (the bi-specific clones) contained fewer mutations on averagethan those that lost HER2 binding. Retaining the Herceptin® antibodyCDR-L3 sequence was preferred but not sufficient to conserve HER2binding. This is consistent with the report that the Herceptin® antibodyCDR-L3 is the most important LC CDR for HER2 binding (Kelley andO'Connell, Biochemistry 32:6828. 1993). Representative VEGF-bindingclones were expressed as Fab and IgG proteins (Table 2).

TABLE 2 The representative antibodies isolated from the light chainlibrary of the Herceptin ® antibody (SEQ ID NOS: 12-23). CDR-L1 CDR-L2CDR-L3 28 29 30 30a 30b 30c 30d 31 32 33 50 51 52 53 91 92 93 93a 93b 94Specificity K_(D)(nM) Herceptin ® D V N — — — — T A V S A S F H Y T THER2 0.1 3-l^(a) N V W D W V P A S S G W Y I A VEGF 15 bH1 D I P R S I SG Y V W G S Y H Y T T VEGF/HER2   300/26 bH3 D I G L G S V W A S Y H Y TT 19,000/8  bH4 D I R S G S V W G S Y H Y T T  3,500/11 ^(a)Differencesfrom Herceptin ® antibody are shown in bold.To demonstrate that these antibodies bound specifically to their cognateantigens and did not interact non-specifically with other proteins, weshowed that there was no detectable binding to a panel of mammalian celllysates and non-antigen proteins. The assay confirmed the mono- andbi-specificity of the purified IgGs or Fabs (FIG. 6).

Equilibrium binding affinities (K_(D)) of the LC library-derivedmono-specific antibodies ranged from 15-150 nM. The bi-specificantibodies bound the new antigens (i.e., VEGF) with high nM to low μMaffinity and HER2 with low nM affinity (Table 2). Of the antibodiesshown in Table 2, the antibody bH1 displayed the highest bi-specificaffinity for the two different protein antigens VEGF (K_(D)=300 nM) andHER2 (K_(D)=26 nM).

Materials

Enzymes and M13-KO7 helper phage were from New England Biolabs. E. coliXL1-Blue was from Stratagene. Bovine serum albumin (BSA), ovalbumin, andTween 20 were from Sigma. Neutravidin, casein, and Superblock were fromPierce Immobilized protein G and anti-M13 conjugated horse-radishperoxidase (HRP) were from GE Healthcare (Piscataway, N.J.). Maxisorpimmunoplates were from NUNC (Roskilde, Denmark). Tetramethylbenzidine(TMB) substrate was from Kirkegaard and Perry Laboratories(Gaithersburg, Md.). All protein antigens were generated by researchgroups at Genentech, Inc. DNA degeneracies were represented using theIUB code and represent equimolar mixtures unless indicated otherwise:N=A/C/G/T, D=A/G/T, V=A/C/G, B=C/G/T, H=A/C/T, K=G/T, M=A/C, R=A/G,S=G/C, W=A/T, Y=C/T.

For example, at certain randomized positions, the wild-type codon wasreplaced by a degenerate NNK codon (N=A/T/G/C, K=G/T in an equimolarratio) that encodes all 20 natural amino acids. The XYZ codon refers toa codon with unequal nucleotide ratios at each position of the codontriplet. X contained 38% G, 19% A, 26% T and 17% C; Y contained 31% G,34% A, 17% T and 18% C; and Z contained 24% G and 76% C.

Phagemid Vectors for Library Construction

Standard molecular biology techniques were used for vector construction.Three templates were constructed for library generation. All templatesare derivatives of plasmid pV0354 used in heavy chain libraries based onmodified humanized 4D5 (version 8) (Lee et al., 2004a).

The 2C4 Fab-C template phagemid pJB0290 was constructed by cloning the2C4 heavy chain variable domain into a pV0354-Fab-C vector containingthe alkaline phosphatase promoter (Lowman et al., 1991) and stIIsecretion signal for both light and heavy chain of Fab. It is engineeredto contain a single cysteine at the C-terminus of the heavy chainvariable domain 1 to allow bivalent M13 phage display of the 2C4 Fab aspreviously described (Lee et al., 2004b). The 2C4 light chain CDRs wereincorporated into the Fab-C vector by site-directed mutagenesis usingthe method of Kunkel et al (Kunkel et al., 1987). An epitope tag (gDtag) (Lasky and Dowbenko, 1984) was added at the C-terminus of the lightchain to enable determination of the level of display as described(Sidhu et al., 2004). The Fab12-G library template pV1283 was created bycloning a highly displayed heavy chain variable domain intopV0354-Fab-C, and the light chain variable domain was modified tocontain CDR-L3 of Fab-12 (humanized A4.6.1, an anti-VEGF antibody). Thehighly-displayed V_(H) was selected from a Fab library that randomizedheavy chain CDR residues of G6 Fab using shotgun alanine scanningmutagenesis (Liang et al., 2006; Vajdos et al., 2002) with CDR-L3converted to Fab-12 (Y₉₁STVPW₉₆; SEQ ID NO:24) by panning on immobilizedanti-gD antibody. The design and construction of the phagemid pV1384,displaying 4d5 (LC-R66G) scFv bivalently on the surface of M13 phageparticles was modified from the template pS2018 described previously(Sidhu et al., 2004). The scFv fragment contained a gD epitope tag inthe linker region between light chain and heavy chain. LC frameworkresidue Arg66 was mutated to G1γ66, which is the prevalent residue inthis position in over 95% of natural kappa light chains. The mutationR66G reduces Herceptin® antibody binding affinity to HER2 only slightly(<2 fold) as described in Kelley and Connell (Biochemistry 32:6828,1993). The CDR sequences of the library templates are summarized in FIG.3.

Library Construction

Phage-displayed libraries were created using oligonucelotide-directedmutagenesis as described (Sidhu et al., 2004). The library templatevectors contained a stop codon (TAA) embedded in CDR-L1, which wasrepaired during the mutagenesis reaction using degenerateoligonucleotides that annealed over the sequences encoding CDR-L1,CDR-L3 (all libraries), CDR-L2 (L1/L2/L3-A, -B, -C, +L4-D) and the lightchain framework 3 (L1/L4 and L1/L2/L3+L4-D). The library mutagenesisreactions were performed according to the method of Kunkel et al (Kunkelet al., 1987). The light chain CDR designs for the libraries aredescribed in FIG. 1, which summarizes the degenerate codons used at eachposition for the different libraries. Three or four oligonucleotideswere mixed at certain ratios for each CDR to encode the desiredfrequency of amino acid types at each position targeted forrandomization (FIG. 4). The oligonucleotides were combined in differentratios to fine-tune the diversity to reflect the amino acid frequency innatural light chain kappa sequences at selected positions. For CDR1,three oligonucleotides containing codons for positions 91-94: CAT NNKNNK RST (SEQ ID NO:25), KMT XYZ XYZ RST (SEQ ID NO:26), or DGG XYZ XYZRST (SEQ ID NO:27) were mixed at 1:3:1 ratios. XYZ is a variation of NNKthat has equal proportions of the A/G/T/C for each site to reduce thecoverage of aliphatic hydrophobic amino acids (Lee et al., J. Mol. Biol.340:1073, 2004). For CDR2, four oligonucleotides containing codons forpositions 50-53: NNK GST TCC NNK (SEQ ID NO:28), TGG GST TCC NNK (SEQ IDNO:29), KGG GST TCC TMT (SEQ ID NO:30), or NNK GST TCC TMT (SEQ IDNO:31) were mixed at 1:1:2:10 ratios. For CDR3, each length was amixture of three oligonucleotides containing codons for position 28-33:G₇₀A₇₀O₇₀ RTT NNK NNK TAC STA (SEQ ID NO:32), G₇₀A₇₀O₇₀ RTT NNK NNK DGGSTA (SEQ ID NO:33), or G₇₀A₇₀C₇₀ RTT NNK NNK NMT STA (SEQ ID NO:34) at1:1:2 ratios. G₇₀A₇₀C₇₀ is a “soft” codon that allows 70% of thedesignated nucleotide and 10% each of the other three, encoding ˜50% ofGlu and ˜50% of the other amino acids.

Structural analysis of a number of representative antibodies with kappaLCs shows that CDR1 has the widest range of conformations, which islikely a result of the variation in loop lengths (11-17 residues betweenposition 24 and 34). Different CDR-L1 lengths (lengths 11-16) were thusincluded in the library. Natural CDR-L3 also varies in length (lengths7-10 residues between position 89-96), which is reflected by the librarydesign (lengths 8-10; FIG. 4).

FIG. 1 shows the comparison of the light chain natural diversity and theactual library designs. The mutagenesis products were pooled into onereaction per library and electroporated into E. Coli SS320 cellssupplemented with KO7 helper phage and were grown overnight at 30° C.(Lee et al., J. Mol. Biol. 340:1073, 2004). ˜10¹¹ cells and ˜5-10 μg DNAwere used in each electroporation reaction. The library phage werepurified (Sidhu et al., J. Mol. Biol. 338:299, 2004). The number oftransformants ranged from 10⁹-10¹⁰. The display level of intact Fabs orscFv on the surface of phage was determined in an ELISA binding assaywhere 96 randomly selected clones from each library were tested fortheir ability to bind an anti-gD antibody. The display level ranged from5-25% (FIG. 2). 25% of the clones displaying antibody retained HER2binding. Approximately 150 displaying clones were sequenced to examinethe actual library diversity as compared to the design diversity. Aportion (˜30%) of the functionally displayed library members retainedthe Herceptin® antibody CDR-L2 and/or CDR-L3 sequence due to incompletemutagenesis (a template stop codon in CDR-1 ensured 100% mutation ofthis CDR in expressed scFvs). These were excluded from the sequenceanalysis of the actual library diversity. At the majority of therandomized positions, the diversity of the phage displayed library ofthe displaying clones did not deviate significantly (p>0.05, odds ratiotest) from the designed diversity. Exceptions were position 29 of theCDR-L1 where Val was found to be slightly over-represented compared toIle (p=0.005) and positions 51 and 53 of CDR-L2, where Gly and Ser weremore prevalent than Ala and Tyr, respectively (p<0.01).

Example 2 Evaluation of Library Performance Library Sorting andScreening

A library was considered functional when antibodies binding specificallyto various protein antigens could be isolated after 4-5 rounds ofsorting. Many protein targets were known to allow functionalimmobilization for library panning and specific antibodies have beengenerated from validated phage-displayed libraries (Fellouse et al.,2005) (Lee et al., 2004a). To evaluate each set of libraries, we chose asubset of these targets for selection (FIG. 2). The libraries weresubjected to an initial round of binding selection with anti-gD antibodyor protein L as the capture target to eliminate clones in which theFab/scFv gene had been deleted, followed by 4-5 rounds of antigenselection. Alternatively, they were directly subjected to target bindingselection without pre-selection with anti-gD or protein L. NUNC 96-wellMaxisorp plates were coated overnight with antigen (5 μg/ml) and blockedfor 1 hour with alternating blocking agents (FIG. 7). Phage solutions of10¹³ phage/ml were added to the coated immunoplates in the firstselection cycle. The phage concentration was decreased in each round ofselection. Following incubation of the phage solutions on theimmunoplates to allow binding to the immobilized antigen, the plateswere washed with PBS, 0.5% Tween 20, repeatedly. To increase thestringency, the incubation time was decreased (4 hours for 1^(st) round,3 hours 2^(nd), 3 hours 3^(d), 2 hours 4^(th), 1.75 hours 5^(th)) andthe number of washes was increased in each round of selection (FIG. 7).Bound phage was eluted with 0.1 M HCl for 30 minutes and the eluant wasneutralized with 1.0 M Tris base. The recovery of phage perantigen-coated immunoplate well was calculated and compared to that of ablocked well without coated antigen to study the enrichment of phageclones displaying Fabs or scFvs that specifically bound the targetantigen (FIG. 7). Eluted phage were amplified in E. coli and used forfurther rounds of selection. Random clones from rounds 4 and 5 wereselected for screening and assayed using phage ELISA in which binding totarget and anti-gD was compared to binding of a non-relevant protein(BSA) for checking non-specific binding. Clones that bound the anti-gDantibody and target but not the non-specific protein were consideredspecific positives. Libraries L1/L3, L1/L4, L1/L2/L3-A, L1/L2/L3-B_(—)1and L1/L2/L3-B_(—)2 did not yield any specific positive clones whereaslibraries L1/L2/L3-C and L1/L2/L3+L4-D enabled isolation of specificantibodies to the target antigens.

For example, random clones from round four were assayed using phageELISA where binding of individually amplified clones to the target andHER2 was compared to binding of a non-target protein (BSA) to testbinding specificity. To enrich the phage clones that maintained HER2binding, the eluted phage from the third and fourth round of VEGF or DR5selection were amplified and subjected to another round of selection onHER2 coated wells. The V_(L) and V_(H) regions of the positive cloneswere amplified by PCR and sequenced.

The hit rate for hFC, hVEGF, and hDR5-lf, was 63, 77, and 85%respectively. The V_(L) regions of the positive clones were amplified byPCR and sequenced as described (Sidhu et al., 2004). The DNA sequenceanalysis of the positive specific binders revealed a percentage ofunique clones of 51% (hFC), 55% (hVEGF), and 6.1% (hDR5-lf). Thesequences of unique hVEGF binding clones are summarized in FIG. 8.

Combined Plate and Solution Selection of hVEGF Binding Clones

High diversity of hVEGF binding clones after four rounds of sorting wasobserved. In order to identify high affinity hVEGF binding clones asolution based selection approach was taken following the 4^(th) platebased sort. 50 nM biotinylated hVEGF was incubated with the phagepropagated from the 4^(th) round of selection on immobilized antigen.After 2 hours of incubation at room temperature with shaking,hVEGF-bound phage was captured on neutravidin-coated and blockedimmunoplates followed by repeated washes. Phage clones were eluted,screened, and sequenced as previously described. Sequences of hVEGFbinding clones from the last solution selection step are found in FIG.9.

Isolation of Bi-Specific Clones from Libraries L1/L2/L3-C andL1/L2/L3+L4-D

The library template for libraries L1/L2/L3-C and L1/L2/L3+L4-D was anscFv fragment modified from the hu4D5 antibody, which binds Her2 withhigh affinity. Mapping of the functional paratope of hu4D5-5 for Her2binding by alanine-scan mutagenesis of the CDR regions showed that heavychain residues contribute the majority of the free energy of binding,whereas individual light chain residues contribute to a lesser extent(Kelley and O'Connell, 1993). Analysis of the atomic structure of theHerceptin® antibody Fab in complex with human Her2-ECD demonstrates thatwhile the light chain is involved in making antigen contact, the heavychain provides most of the structural interface with the antigen (Cho etal., Nature 421:756, 2003). We observed that some members of thefunctional light chain libraries built upon Herceptin® antibody templateretained Her2 binding ability. In an attempt to isolate bi-specific scFvfragments from the functional libraries L1/L2/L3-C and L1/L2/L3+L4-D,capable of binding Her2 as well as a second antigen, two strategies wereapplied. In one approach the positive clones from the previouslydescribed target antigen selection was screened by ELISA for ones thatretained Her2 binding. The percentage of specific positive clonescapable of binding Her2 varied depending on the second antigenspecificity. Only 1 out of 61 unique hFc specific positive clones clonestill bound Her2 (1.6%), 30 out of 41 unique hVEGF binding clones stillbound Her2 (73%), and 2 out of 5 unique hDR5 binders still bound Her2(40%). In addition, a selection-based approach was taken to isolatebi-specific antibodies by selecting Her2 binders from the pool of hVEGFand hDR5 binding antibodies. The elution from round 4 of target antigensorting was subjected to an additional round of selection by incubating2×10¹³ phage/ml on Her2 coated (5 μg/ml) and BSA-blocked Maxisorpimmunoplates for 1 hour. The plates were washed 15 times with PBS, 0.5%Tween 20 and bound phage eluted as described previously. Random cloneswere selected and assayed for Her2, anti-gD and target binding andcompared to non-specific binding to an un-relevant protein (BSA). All192 clones tested were identified as specific positives and sequenced asdescribed previously. Sequencing revealed 94 unique sequences. Insummary, this method generated 94 Her2/hVEGF bi-specific clones out ofthe 94 unique clones tested (100%) (FIG. 8). The sequences of allisolated unique hVEGF/Her2 bi-specific antibodies from both isolationstrategies are summarized in FIGS. 10A and 10B. The sequences ofisolated clones that lost all detectable binding to Her2 are shown inFIG. 11. Of the clones that have dual specificity, nearly all retainedthe Herceptin® antibody CDR-L3, making it likely that maintaining CDR-L3is important for maintaining HER2 binding. In the case of hDR5, 2 out ofthe 7 unique Her2-binding clones were bi-specific (29%, 12 clonessequenced). One of the dual specific clones had some homologous changesin CDR-L3.

High-Throughput Characterization of hVEGF Binding Clones

A high-throughput single spot competitive ELISA in a 96-well format(Sidhu et al., 2004) was used to screen for high affinity clones forhVEGF and to study the VEGFR1-blocking profiles. Briefly, MaxisorpImmunoplates were coated with 2 μg/ml hVEGF₁₀₉, overnight at 4° C. andblocked with 1% (w/v) BSA for 1 hour. Phagemid clones in E. coliXL1-Blue were grown in 150 μl of 2YT broth supplemented withcarbenicillin and M13-KO7 helper phage; the cultures were grown withshaking overnight at 37° C. in a 96-well format. Culture supernatantscontaining phage were diluted five-fold in PBST (PBS with 0.05% Tween 20and 0.5% (w/v) BSA) with or without the addition of 100 nM hVEGF₁₀₉ foraffinity screen. For receptor blocking screens, hVEGF coated wells wereincubated with or without VEGFR1 Domain 1-3 (D1-3) and VEGFR1 Domain 2(D2) before adding five-fold diluted phage supernatant (Liang et al.,2006; Wiesmann et al., 1997). After incubation for 1 hour at roomtemperature (RT), the mixtures were transferred to the coated plateswith hVEGF₁₀₉ and incubated for 10 minutes. The plate was washed withPBT (PBS with 0.05% Tween 20) and incubated for 30 minutes with anti-M13antibody horseradish peroxidase conjugate diluted 5000-fold to 1 nM inPBST. The plates were washed, developed with TMB substrate forapproximately five minutes, quenched with 1.0 M H₃PO₄, and readspectrophotometrically at 450 nm. In the single-spot affinity assay, theratio of the absorbance in the presence of solution-phase hVEGF₁₀₉ tothat in the absence of solution-phase hVEGF₁₀₉ was used as an indicationof the affinity. A low ratio would indicate that most of the Fab-phagewere bound to solution-phase hVEGF₁₀₉ in the initial incubation stageand, therefore, were unavailable for capture by immobilized hVEGF₁₀₉.The high-throughput affinity assay results of the first 41 unique clonesare summarized in FIG. 12. Similarly, for the blocking assay, a lowratio indicated that the binding of a clone to hVEGF₁₀₉ is blocked bythe hVEGF₁₀₉-VEGFR1 interaction, indicating that some clones have anoverlapping binding site (epitope) on VEGF with the respective VEGFreceptor fragments (FIGS. 13A and 13B) and these clones are likely to bedisplaying the blocking antibodies.

High-Throughput Characterization of Bi-Specific hVEGF/Her2 Clones

The same principle as described in the previous section was applied toenable isolation of clones with high affinity for hVEGF and Her2 forfurther characterization (FIG. 14A). The high-throughput single pointcompetitive ELISA was used to screen for high affinity clones for hVEGFand Her2 by coating Maxisorp Immunoplates with 2 μg/ml hVEGF₁₀₉, andHer2-ECD overnight at 4° C., followed by blocking with 1% (w/v) BSA for1 hour. Phage clones that were identified as bi-specific in the previoussingle spot ELISA screen were grown as described previously andincubated with and without the addition of 20 nM Her2-ECD and 50 nMhVEGF. After incubation for 1 hour at room temperature, the solutionswere applied to the coated immunoplates and the binding signals recordedand analyzed as described in the previous section. Clones with low ratiofor both hVEGF and Her2 were selected for further characterization.hVEGF-specific and hVEGF/Her2 bi-specific phage clones that gave rise tothe lowest signal ratios in the single spot competitive ELISA wereselected for affinity measurement by competitive ELISA as well as theDR5-binding and DR5/Her2 bi-specific phage clones from the initialsingle spot ELISA screen and VEGF binding clones from the combined plateand solution selection. Phage clones were propagated from a singlecolony by growing in 25 ml of 2YT culture supplemented withcarbenicillin and KO7 helper phage overnight at 30° C. Phage purified byprecipitation in PEG/NaCl were first diluted serially in PBST and testedfor binding to an antigen-coated plate. The dilution that gave 50-70%saturating signal was used in the solution binding assay in which phagewere first incubated with increasing concentration of antigen for one totwo hours and then transferred to antigen-coated plates for 10-15minutes to capture the unbound phage. IC₅₀ was calculated as theconcentration of antigen in solution-binding stage that inhibited 50% ofthe phage from binding to immobilized antigen (Lee et al., 2004a). FIG.14B depicts the curves from which the IC₅₀ was calculated for theanalyzed hVEGF binding clones from the plate sorting strategy. The IC₅₀values ranged from 22 nM to >1 μM (FIG. 14B). The IC₅₀ values for thehVEGF binders isolated by combined plate and solution based selectionranged from 41 nM-226 nM (FIG. 9). IC₅₀ values of DR5-binding clonesranged from 20 nM to >1 μM. The IC₅₀ values for hVEGF/Her2 bi-specificclones are summarized in FIG. 15.

Example 3 Characterization of Antibodies from the Light Chain Library

Conversion of scFvs to Fabs

To test whether conversion of the scFvs′2 as displayed on phage to Fabsaffected the affinity of the binding clones from the library, 2 clones(3-7 anti-hVEGF and 4-1 anti-hDR5) were chosen for conversion to Fab anddisplayed on phage. The V_(L) region of phagemid DNA for selected hVEGFand DR5 scFv fragments was digested with restriction enzymes, whichcleaved the DNA upstream of the region encoding for CDR-L1 (EcoRV) anddownstream of the region encoding for CDR-L3 (KpnI). The digested DNAfragment was ligated into a similarly digested vector (pAP2009) designedfor the phage display of Fab hu4D5 by fusion to the C-terminal domain ofthe M13 gene-3 minor coat protein (Lee et al., 2004b). The resultingbi-cistronic phagemid contains the light chain fused to an epitope (gD)tag at the C-terminus and heavy chain (V_(H) and C_(H)1) fused to thegene for M13 minor coat protein (p3) C-terminally under the control ofthe alkaline phosphatase promoter. The first open reading frame encodeda polypeptide consisting of the stII secretion signal followed by theFab4D5 light chain, with the CDRs replaced by those of 3-7 anti-hVEGFand 4-1 anti-hDR5 scFv′2, followed by a gD-tag epitope. The second openreading frame encoded a fusion polypeptide consisting of the following:the stII secretion signal, the Fab4D5 heavy chain, an amber (TAG) stopcodon, a Gly/Ser linker sequence and c-terminal domain of g3 protein(cP3). Expression in E. coli XL-1 Blue co-infected with M13-KO7 resultedin the production of M13 bacteriophage displaying Fab versions of 3-7and 4-1 scFv′2. Competitive phage ELISAs were used to estimate theaffinities of the phage-displayed scFvs and Fabs for hVEGF and hDR5 asIC₅₀ values. The data from the two different formats were in goodagreement (data not shown).

To enable display of bH1 Fab on the surface of M13 bacteriophage,plasmid pAP2009 was modified to encode bH1Fab. Versions of the bH1 Fabwere used as library templates containing stop codons (TAA) in eitherthe three LC CDRs or the three HC CDRs for the LC and HC library,respectively. Separate heavy chain and light chain alanine and homologscanning libraries were constructed as previously described (Vajdos etal., J. Mol. Biol. 320:415, 2002). The degeneracy ranged from 1×10⁵ to1×10⁸ and the actual library size from 6×10⁹ to 4×10¹⁰. The librarieswere constructed as described above. Two to three rounds of selectionwere performed on immobilized targets (VEGF, HER2-ECD, protein L, oranti-gD mIgG) (Vajdos et al., J. Mol. Biol. 320:415, 2002). Targetbinding clones were screened by phage ELISA for target binding followedby DNA sequencing and sequence alignment to calculate thewild-type/mutation ratios at each position. The ratios from sequenceanalysis of approximately 100 unique sequences of VEGF and HER2 bindingclones were corrected for display and protein folding effect by dividingwith ratios calculated from the sequences of more than 100 anti-gDbinding clones to yield the F_(wt/mut) values. As only the Fab heavychain is fused to the phage coat, the phage display of the gD tag, whichis fused to the light chain, is indicative of proper folding andassociation of light chain and heavy chain. Consistently, protein Lbinding to a non-linear epitope on the light chain of the Fab alsoresulted in similar wild-type/mutation ratios as gD tag selections.F_(wt/mut) values were converted to ΔΔG using the formula ΔΔG=RTln(K_(a,wt)/K_(a,mut))=RT ln(F_(wt/mut)) as described in Vajdos et al.(J. Mol. Biol. 320:415, 2002).

Expression of Library Binders as Free Human Fab and IgG

To accurately determine the affinity, specificity and other propertiesof the antibodies, representative clones from each specificity groupexhibiting the highest affinity in the competition ELISA experimentswere selected for expression as free Fab and hIgG (FIG. 16). Thevariable domain of light chain and heavy chain was cloned into a vectorpreviously designed for Fab expression in E. coli or transient human IgGexpression in mammalian cells (Lee et al., 2004a). Fab protein wasgenerated by growing the transformed 34B8 E. coli cells in completeC.R.A.P. medium at 30° C. for 26 hours as described (Presta et al.,1997). The hIgGs were expressed by transient transfection of 293 cellsand hIgG was purified with protein A affinity chromatography (Fuh etal., J. Biol. Chem. 273:11197, 1998). The 1 L E. coli cultures werepurified with protein G affinity chromatography. The columns were washedwith PBS and Fab protein was eluted with 100 mM acetic acid and dialyzedagainst PBS. The 4 L E. coli cultures were purified on a protein Aaffinity column followed by cation exchange chromatography as previouslydescribed (Muller et al., 1998). Protein concentrations were determinedspectrophotometrically. The final yield for Fab was typically 0.8-15mg/1 purified from a small-scale shake flask growth. IgG productionyield was medium to high at 6.7-60 mg/1 in small-scale culture (FIG.17). The purified proteins were first characterized using size exclusionchromatography and light scattering to ensure that the proteins did notexhibit significant levels of protein aggregation (<5%).

Briefly, the Fabs and hIgGs expressed were screened by ELISA for bindingtheir respective antigen(s). All but one variant were found to bindtheir cognate antigen(s). Clone 4-6 lost hDR5 binding ability whenconverted to Fab and hIgG. Selected anti-VEGF clones, raised against theshorter form hVEGF₁₀₉, were tested for binding to hVEGF₁₆₅ usingstandard ELISA (H3, H4_N, H4_D hIgG), and competitive ELISA (bH1, 3-1,3-6, 3-7 hIgG). G6 hIgG (Fuh et al., 2006) was used as a positivecontrol (FIGS. 18A and 18B). As expected, all clones bound hVEGF₁₆₅.

To study the extent of protein aggregation selected clones were analyzedby Size-Exclusion chromatography (SEC) followed by Light Scattering (LS)Analysis as purified Fab and IgG. The samples were assayed in PBS at aconcentration of 0.5 mg/ml (hIgG) and 1 mg/ml (Fab). A maximum of 5%aggregation was observed for all samples at the given concentration(FIG. 17), which is within range of what we have previously observed forother phage-display derived antibodies. Clones 3-6 and 3-7 did not comeout at the expected time point, which suggested these reformatted IgGand Fab exhibit aggregation and or non-specific interaction with theresin (data not shown). These clones were taken out of the set of clonesthat underwent further analysis.

To rule out cross-reactivity and non-specific binding, we studiedbinding of selected hIgG at high concentration (100 nM) to a panel ofimmobilized a panel of protein targets including whole cell lysates, thecognate antigens, and homologues in a standard ELISA assay. In additionto antigen, we immobilized a murine version of hVEGF to testcross-species reactivity of the anti-hVEGF clones. In particular, thepanel of proteins was immobilized on Maxisorp plates and blocked with 1%BSA in PBS for 1 hour. The hIgGs (or Fabs) were diluted in PBST to aconcentration of 100 or 500 nM and transferred to the coated plates.After a 1-hour incubation, the plates were washed and incubated withHRP-conjugated protein A. The binding signals were developed by additionof TMB substrate for approximately 5 minutes, quenched with 1M H₃PO₄,and read spectrophotometrically at A₄₅₀. The hIgGs tested boundspecifically to their antigen(s). Clones bH1 and 3-1 exhibitedcross-reactivity to murine VEGF (mVEGF) (FIG. 19).

To test whether the bi-specific antibodies bH1, H3 (anti-hVEGF/Her2),and D1 (anti-hDR5/Her2) could simultaneously bind their cognate antigensor if the antigens compete for antibody binding, hVEGF and hDR5 wereimmobilized at a concentration of 2 μg/ml. A fixed concentration of hIgGwas incubated with serial dilutions of Her2-ECD followed by capture ofthe hIgG on the immobilized antigen. In each case, Her2-ECD binding wasfound competitive with binding to the other antigens (FIG. 20).

To accurately determine the affinity of IgGs and Fabs (i.e., anti-hVEGFand anti-hVEGF/Her2 Fab and IgG isolated from the libraries) and tostudy the binding profiles in real time, we used surface plasmonresonance (SPR) assays on a BIAcore™-3000 (BIAcore, Uppsala, Sweden)machine with immobilized hVEGF, mVEGF, DR5, and Her2-ECD CM5 sensorchips at response units (RU) of 40-300 depending on the analyte studied.Immobilization was performed as described (Chen et al., 1999). Tominimize avidity effects of the bivalent IgG analytes, a lower densityof ligand was targeted on the sensor chip in these cases. Samples ofincreasing concentrations ranging from a concentration approximately10-fold below to 10-fold above the estimated K_(D) (based on competitionELISA experiments) were injected at 22-30 μl/minute, and bindingresponses were corrected by subtraction of RU from a referenceflow-cell. In addition, the responses were double referenced tonormalize for instrument drift by subtracting RU from ligand-conjugatedflow-cell injected with sample buffer (PBS with 0.05% Tween 20). Forkinetic analysis of the Fabs, a 1:1 Langmuir binding model of was usedto calculate the k_(on) and k_(off). When necessary (at high analyteconcentrations) a 1:1 Langmuir binding model with mass-transferlimitation was applied. For the IgG analytes, a bivalent analyte bindingmodel with or without mass-transfer limitation was used (BIAcoreEvaluation Software 3.2). In the case of H3 hIgG, H4_N Fab, and H4_DhIgG, the fit of responses to the kinetic binding models was notsatisfactory. Therefore, steady state binding analysis was applied wherethe equilibrium response was plotted against analyte concentration. TheK_(D) was estimated as the EC₅₀. A summary of the BIAcore bindinganalysis can be found in FIG. 21. The affinity of the hVEGF bindingantibodies 3-1, 3-6 and 3-7 was found to be in the nano molar range. Thebi-specific antibodies analyzed (bH1, H3, H4_N, H4_D) showed lowmicromolar to micromolar affinities for hVEGF. In contrast, theaffinities for Her2 ranged from 8-59 nM (Fab).

To determine whether the light chain of anti-hVEGF binders bH1, H3, andH4_N could bind hVEGF independent of the sequence of the associatedheavy chain, the light chain variable domains were grafted onto theanti-Her2 2C4 Fab by cloning the light chain variable domains into a 2C4Fab expression vector pJB0524, thus replacing 2C4 light chain variabledomain. The Fabs were expressed as previously described. The bH1/2C4 andH3/2C4 chimeric Fabs did not express at detectable levels. The H4_N/2C4chimeric Fab protein was isolated and tested for binding to hVEGF (bH1original specificity) and Her2 (bH1, 2C4 original specificity). Nobinding was detected to hVEGF and Her2 by a standard ELISA binding assay(FIG. 22). The results indicate that the heavy chain of bH1 is requiredfor antigen binding.

Comparison of Anti-hVEGF Epitopes

In an attempt to roughly map out the epitopes of the anti-hVEGFantibodies on hVEGF, we studied the ability of these newly isolatedanti-VEGF antibody to compete with other hVEGF binding antibodies andVEGF receptors with known binding sites (Fuh et al., 2006; Muller etal., 1998; Wiesmann et al., 1997). The assays were done in a competitiveELISA format where the VEGFR1 (Flt) Domain 1-3 and anti-hVEGF antibodiesAvastin® (IgG), B20-4.1 (IgG), G6 (Fab), and KDR Domain 1-7 Fc fusionprotein were immobilized on Maxisorp immunoplates at 2 μg/ml. Thesolution competition binding assay used biotinylated VEGF equilibratedwith serial dilutions of purified IgG proteins, and the unboundbiotin-VEGF was captured with immobilized Fab or IgG coated on Maxisorbplates and was detected with streptavidin-conjugated HRP (Lee et al., J.Mol. Biol. 340:1073, 2004). Antibodies that block hVEGF from bindingother hVEGF-binding antibodies or hVEGF-receptors are likely to shareover-lapping epitopes. High concentrations (μM) of the bi-specifichVEGF/Her2-binding antibody, bH1, enabled complete blocking of hVEGFbinding to its receptors, VEGFR1 and VEGFR2, suggesting bH1 epitopeoverlaps sufficiently with VEGFR1 (FIG. 23) and VEGFR2 (FIG. 23). Inaddition, bH1 blocks hVEGF binding to B20-4.1 (FIG. 24). H3, H4_N, andH4_D also block hVEGF-binding to both receptors, which points to similarepitopes as bH1 (FIG. 23). The incomplete blocking profiles are likelyto be a consequence of their relatively low affinity for hVEGF (FIG.21). 3-1, in contrast, does not block hVEGF from binding VEGFR1, even atthe highest concentration (0.5 μM) (FIG. 23). Furthermore, we could notdetect 3-1 hIgG blocking of the Avastin® antibody (FIG. 25). However,3-1 hIgG block hVEGF binding to VEGFR2 (KDR) (FIG. 23) as well as toB20-4.1 (FIG. 24). These results indicate that 3-1 has a unique epitopecompared to the other antibodies.

Example 4 Structure-Function Studies of bH1, Anti-hVEGF/her2 Bi-SpecificAntibody

To elucidate the nature of the bH1 interaction with its two antigens,VEGF and HER2, structural and functional studies was performed. TheHerceptin® antibody and bH1 differ in CDR-L1 (V²⁹NTA³² vs. I²⁹PRSISGY³²;SEQ ID NOS:35 and 36) and CDR-L2 (S⁵⁰ASF⁵³ vs. W⁵⁰GSY⁵³; SEQ ID NOS:37and 38). The bH1 anti-VEGF/Her2 was chosen as representative forstructural characterization based on its dual specific nature and itsrelatively high affinity for VEGF and Her2. In order to study thefunctional and structural epitopes on VEGF and Her2, we crystallized thebH1 Fab in complex with VEGF₁₀₉ and the extracellular domain of hHer2and solved the structures of the two complexes by X-ray crystallography.In addition, we performed alanine and homolog shotgun scanning analysisusing combinatorial phage displayed libraries as described (Vajdos etal., 2002).

bH1 Fab Expression, Purification, Crystallization and Data Collection

The receptor-binding portion of human VEGF, consisting of residues8-109, was expressed, refolded and purified as described previously(Christinger et al., 1996). Residue 1-624 of the extra cellular domainof Her2 was expressed and purified as previously described (Franklin etal., 2004; Hudziak and Ullrich, 1991).

For large-scale bH1 Fab preparation, whole cell pellet was obtained froma ten liter E. coli fermentation. 220 grams of cell paste was thawedinto 1 L PBS, 25 mM EDTA, 1 mM PMSF. The mixture was homogenized andthen passed twice through a microfluidizer. The suspension was thencentrifuged at 12 k in 250 ml aliquots for 90 minutes. The protein wasthen loaded onto a Protein G column (25 ml) equilibrated with PBS at 5ml/minute. The column was washed with equilibration buffer and theneluted with 0.58% acetic acid. The fractions were assayed by SDS PAGE(data not shown). Fractions containing bH1 Fab were pooled and thenloaded onto a 50 ml Cation Exchange SP Sepharose column (Pharmacia)equilibrated with 20 mM MES pH 5.5. The Fab was eluted with a sodiumchloride gradient in the equilibration buffer. The gradient was linearto 0.5 M NaCl, 20 mM MES pH 5.5. Fractions containing the Fab wereidentified by SDS-PAGE (data not shown), and pooled. bH1 Fab eluted at aNaCl concentration of approximately 0.5 M. The Fab concentration wasdetermined by measuring the A₂₈₀. The final yield for bH1 Fab was 67mg/1 fermenter growth.

Complexes were obtained by mixing the purified bH1 Fab and VEGF or Her2ECD in 2:1 molar ratio and purified by size-exclusion chromatography(SP-200, Pharmacia) in 25 mM Tris-HCl, pH 7.5 and 0.3 M sodium chloridefor VEGF-Fab complex and with 25 mM Tris-HCl, pH 8 and 0.15 M sodiumchloride for the Her2 ECD-Fab complex. The composition of the resultingcomplexes was verified by SDS PAGE (data not shown). The protein complexwas concentrated and used in crystallization trials. Initialhanging-drop experiments using the vapor-diffusion method at 19° C.resulted in small isomorphous crystals from 14 different conditionswithin 1 week in the case of the bH1-VEGF complex. Crystals of thebH1-Her2 complex appeared in 4 conditions within a week. Crystals fromone condition was chosen for further optimization in each case.

For crystallization of bH1 Fab-VEGF (8-109), equal volumes of proteincomplex solution (10.6 mg/ml protein, 300 mM NaCl, 25 mM Tris-HCl pH7.5) and crystallization buffer containing 0.15 M D, L Malic Acid pH7.0, 20% PEG₃₃₅₀ was mixed and equilibrated at 19° C. Large crystalsappeared after 24 hours which belonged to space group C222₁ with celldimensions of a=100.6, b=198.0, c=77.7. The crystal forms contained 1Fab and 1 VEGF monomer in the asymmetric unit. Prior to data collectionthe crystals were cryo-protected by transfer between drops containing5%, 10%, and 15% glycerol in artificial mother liquor, followed by aflash freeze in liquid nitrogen. Data was collected to 2.6 Å at the beamline 5.0.1 of the Advanced Light Source (Berkeley).

Crystals of bH1 Fab-Her2(1-624) was obtained by mixing protein solution(11 mg/ml, 25 mM Tris pH 8 and 150 mM sodium chloride) withcrystallization buffer containing 25% w/v PEG₂₀₀₀, 0.1M MES pH 6.5.Crystals appeared after 12 hours that belonged to space group P2₁2₁2₁with cell dimensions of a=62.3, b=115.1, c=208.2. The crystals containedone Her2-Fab complex in the asymmetric unit. Before data collection thecrystals were flash frozen in liquid nitrogen with 20% Ethylene Glycolas cryo-protectant. Data was collected to 2.9 Å at the beam line 5.0.1of the Advanced Light Source (Berkeley).

Data Processing, Structure Determination, and Refinement

The data was processed using Denzo and Scalepack (Otwinowski, 1997). Thestructures of bH1 Fab complexes was solved by Phaser (L. C. Storoni,2004; Read, 2001). The bH1-Fab-VEGF(8-109) complex was solved usingcoordinates of VEGF from a previously described VEGF-Fab complex (2FJG)and Fab fragments containing either the variable domains V_(L)/V_(H) orthe constant domains C_(H)/C_(L) of the Herceptin® antibody Fab-Her2complex (1N8Z). Fragments of Her2 and the variable domain of theHerceptin® antibody Fab from the Her2-Fab complex 1N8Z were used assearch models when solving bH1-Her2 structure. The constant domain ofthe bH1 Fab could not be found using the Herceptin® antibody Fabconstant portion as a search model (1N8Z) and had to be docked manuallyguided by the Herceptin® antibody Fab-Her2 complex structure. Modelbuilding and refinement were performed using the programs Refmac(Collaborative Computational Project, 1994) and Coot (Emsley and Cowtan,2004), respectively. Stereochemical parameters were analyzed usingMolProbity (Lovell et al., Proteins 50:437 (2003)). The structures wererefined to R_(value)=0.22 and R_(free)=0.27 for the Fab-VEGF-complex andR_(value)=0.25 and R_(free)=0.31 for the Fab-Her2-complex. A crystalstructure of bH1 Fab in complex with VEGF as well as Her2-ECD wasmodeled. Some bH1 Fab residues were within 4.5, 4.0, and 3.5 Å of theantigens. The two paratopes (the area on the antibody that makes contactwith the antigen) for the two antigens on the same antibody overlapsignificantly and residues from both light chain and heavy chain areinvolved with the binding with both antigens. bH1 binds a similarepitope on VEGF as the Avastin® antibody, and bH1 binds Her2 on anessentially identical epitope as the Herceptin® antibody.

The crystal structures of bH1 Fab bound to the extracellular domain(ECD) of HER2 (residue 1-624) and to the VEGF receptor-binding domain(residue 8-109) were determined at 2.9 Å and 2.6 Å resolutions,respectively (FIG. 26 and Table 3). FIG. 26 shows the bH1 Fab/HER2crystal structure superimposed with the Herceptin® antibody/HER2complex, and the crystal structure of the bH1 Fab/VEGF complex.

TABLE 3 Crystallographic Studies bH1 Fab/hVEGF bH1 Fab/HER2- complex ECDcomplex Data Collection Statistics Space group C222₁ P2₁2₁2₁ Unit Cell(Å) a = 100.6, b = a = 62.3, b = 198.0, c = 77.7 115.1, c = 208.2Beamline, wavelength ALS 5.0.1 ALS 5.0.1 Resolution (Å) 50.0-2.650.0-2.9 Rsym^(a) 0.090 (0.66) 0.095 (0.66) Number of Observations151689 192951 Unique Reflections 24705 34149 Completeness (%)*  99.8(100)  100 (100) I/σ (I)* 16.0 (3.0) 18.5 (2.6) Refinement StatisticsContent of assymmetric unit 1/2 VEGF 1 Her2-ECD dimer, 1 Fab monomer, 1Fab Resolution (Å) 30.0-2.6 30.0-2.9 Reflection used 22977 32277 RFactor^(b), Rfree 0.19, 0.25 0.22, 0.28 RMS Deviation Bonds (Å) 0.0110.010 RMS Deviation Angles (°) 1.3 1.3 Ramachandran Statistics FavouredRegions (%) 96.5% 89.9% Allowed Regions (%) 99.4% 97.9% Outliers (%)0.6% 2.1% Number of Residues 528 1017 Numbers of waters 49 0 Number ofSugars 0 2 Number of Ligands/Ions 1 (Glycerol) 1 (MES) Rsym^(a) = Σ I −<I> ΣI. <I> is the average intensity of symmetry-related observations ofa unique reflection. R Factor^(b) = Σ F0 − Fc ΣI F0. Rfree is calculatedas R except for 5% of the reflections excluded from all refinements.*Values in parenthesis denote values of the highest resolution shell.

In the bH1/HER2 complex, the Fab binds to domain IV of HER2 in a mannersimilar to the Herceptin® antibody (Cho et al., Nature 421:756, 2003);the two complexes superimpose with a root mean square deviation(r.m.s.d.) of Cα positions of 2.3 Å. In the VEGF complex, bH1 recognizesan epitope that overlaps with the binding sites of the VEGF receptorsVEGFR1 and VEGFR2 and of other VEGF antibodies (Wiesmann et al., Cell91:695, 1997; Muller et al., Proc. Natl. Acad. Sci. USA 94:7192, 1997).Consistently, the bH1 blocking of VEGF binding to its receptors wasobserved (FIG. 27). For the data shown in FIG. 27, biotinylated humanVEGF₁₆₅ was equilibrated with increasing concentrations of IgG (x-axis).Unbound hVEGF₁₆₅ was captured on immobilized VEGFR2-ECD Fc fusion anddetected spectrophotometrically (optical density at 450 nm, y-axis).

As shown in FIG. 28, the binding sites for VEGF and HER2 on bH1 overlapextensively. Twelve out of the fourteen residues that engage HER2 alsocontact VEGF. Both binding sites include CDR residues from the HC aswell as LC. In the HER2 complex, the LC and HC CDRs contributeapproximately equal antigen contact area (53% and 47% respectively)while in the VEGF complex, the LC CDRs constitute nearly 70% of theburied surface (FIG. 29). The HER2 binding site on the Herceptin®antibody and bH1 are similar and differ only in the CDR-L1 and -L2regions where the Herceptin® antibody sequence is not conserved in bH1(FIG. 28). In FIG. 28, residues on the bH1 or the Herceptin® antibodyFab surface are shaded according to the extent buried by VEGF or HER2(dark shading and white lettering >75% buried, intermediate shading andwhite lettering 50-75% buried, light shading and black lettering 25-49%buried). The underlined residues differ between bH1 and the Herceptin®antibody. The white dotted line depicts the divide of light and heavychain.

The conformation of bH1 Fab in complex with HER2 is markedly similar tothat of the VEGF-bound Fab (r.m.s.d.=0.7 Å, Cα). The CDRs of both bH1Fab structures superimpose well with each other and with the parentHerceptin® antibody Fv and bH1 Fv (HER2) r.m.s.d.=0.6 Å, the Herceptin®antibody Fv and bH1 Fv (VEGF) r.m.s.d.=1.2 Å. The CDR-L1 is an exceptionand differs significantly in the two complex structures; the deviationis 4.6 Å (Cα of residues 27-32). FIG. 30 shows that the CDRconformations of bH1 Fab bound to VEGF are markedly similar toHER2-bound bH1 and to the Herceptin® antibody, with exception of theCDR-L1. FIG. 30 is a superposition of the CDR loops as tubes ofVEGF-bound bH1 (dark shading), HER2-bound bH1 (white) and HER2-bound theHerceptin® antibody (light shading). The CDR-L1 loop exhibitssignificantly different conformations in the two bH1 structures(r.m.s.d._(Cα)=4.6 for bH1 residues 27-32) (FIG. 31). In the HER2complex, the CDR-L1 is minimally involved in antigen interaction andpart of the loop (residues 28-30b) appears flexible. For VEGF binding,the entire loop is well structured and contributes 26% of surface areaburied by VEGF.

Two residues in CDR-L1, Ile30c and Tyr32, have different conformationsand play different roles in bH1 binding to HER2 or VEGF. In the HER2complex, the side chain of Ile30c is buried in the hydrophobic coreformed by CDR-L1 and CDR-L3 residues. In the VEGF complex, this sidechain forms hydrophobic contacts with VEGF. The Cα of Tyr32 is in thesame position in the two structures, but its side chain is rotated ˜130degrees. In the HER2 complex Tyr32 packs against the receptor, while inthe VEGF complex the side chain, together with Ile29, form thehydrophobic core and support the conformation of CDR-L1 and CDR-L3. TheCDR-L1 conformation is further stabilized by hydrogen bonds betweenTyr32 and the LC framework residue G1γ72. The structural analysisconfirms that Tyr32 is critical for VEGF binding as mutation to eitheralanine or phenylalanine is not tolerated. Contrary to VEGF binding,mutation of Tyr32 to alanine (back to the Herceptin® antibody residue)is preferred for HER2 binding. Superposition of the two complexesreveals that VEGF would clash with Tyr32 of CDR-L1 in its HER2 boundstate (FIG. 31). In FIG. 31 the side chains of residues Tyr32, Ile30c,Ile29, and G1γ72 are shown as sticks. Residues with temperature factorshigher than average are shown in darker shading (residues 28-30b).Hydrogen bonding between Tyr32 and G1γ72 is illustrated by a dottedline.

The above results indicate that the capability to rearrange CDR-L1 isnecessary for the bi-specificity of bH1 Similar conformationalflexibility of CDR-L1 has been shown to play a role in antigenrecognition of natural antibodies (Jimenez et al., Proc. Natl. Acad.Sci. USA 100:92, 2003; Mylvaganam et al., J. Mol. Biol. 281:301, 1998).FIGS. 26, 28, 30, 31, and 32 are generated from the crystal structurecoordinates (PDB codes, 3BDY, 3BE1, 1N8Z) using PYMOL (DeLano Scientfic,San Carlos, Calif.).

bH1 Shotgun Scanning

To study the antigen-binding sites of bH1Fab, shotgun scanningcombinatorial mutagenesis using phage-displayed Fab libraries wasperformed (Vajdos et al., J. Mol. Biol. 320:415, 2002; Weiss et al.,Proc. Natl. Acad. Sci. USA 97:8950, 2000). Binding selections on theantigens (hVEGF and Her2-ECD) to isolate functional clones followed byDNA sequencing enabled calculations of wild-type/mutant ratios at eachvaried position (Vajdos et al., 2002). These ratios were then used todetermine the contribution of each scanned side-chain to VEGF and Her2binding. The results enabled mapping of the functional paratope forbinding VEGF and Her2.

bH1 Shotgun Library Design

Solvent exposed residues in the CDRs were scanned using phage-displayedlibraries in which the wild type residues were allowed to vary as eitheralanine or wild type (Alanine Scan) or as a homolog residue or wild type(Homolog Scan). The nature of the genetic code required some othersubstitutions to be included in the library in addition to Wt/Alanine orWt/Homlog residues (FIG. 33). Separate heavy chain and light chainalanine and homolog scanning libraries were constructed. The librariesare described in FIG. 34. The degeneracy ranged from 1.3×10⁵ to 1.3×10⁸and the actual library size from 6×10⁹ to 4×10¹⁰.

Construction of Shotgun Scanning Libraries

As noted above, to enable display of bH1 Fab on the surface of M13bacteriophage, a previously described plasmid AP2009 designed to displayhu4D5Fab on phage fused to the C-terminal domain of the M13 gene-3 minorcoat protein, was modified to encode bH1Fab using standard molecularbiology techniques. The C-terminus of the light chain contained anepitope (gD) tag. “Stop template” versions of the bH1 Fab was used aslibrary template (Sidhu et al., 2004). The light chain alanine andhomolog scanning library had stop codons in CDR-L1, CDR-L2 and CDR-L3and the heavy chain alanine and homolog libraries contained stop codonsin each heavy chain CDR. The libraries were constructed by previouslydescribed methods (Sidhu et al., 2004) using Kunkel mutagenesis (Kunkelet al., 1987) on the respective stop templates.

Library Selection

NUNC 96-well Maxisorp immunoplates were coated with 5 μg/ml capturetarget (hVEGF₁₀₉, Her2-ECD or anti-gD mIgG) and blocked with 1% BSA(w/v) in PBS. Phage from the above-described libraries were propagatedwith KO7 helper phage (NEB) as described (Lee et al., 2004a). Thelibrary phage solutions were added to the coated plates at aconcentration of 10¹³ phage particles/ml and incubated for 1-2 hours atRT. The plates were washed 8 times with PBST and followed by elution ofbound phage with 0.1 M HCl for 30 minutes. Enrichment after each roundof selection was determined as described previously. After 2 rounds oftarget selection, 50-1000-fold enrichment was observed for all librariesexcept LC-Ala and LC-Hom sorted on hVEGF, which showed 5-10-foldenrichment. A number of random clones from each library exhibiting50-1000-fold enrichment was selected for sequencing as described (Sidhuet al., 2004). Library LC-Ala was screened for hVEGF binding in phageELISA (Sidhu et al., 2000). Clones that exhibited hVEGF ELISA signals atleast two-fold over signals on a control plates coated with BSA wereselected for sequencing. The LC-Hom library was subjected to 1additional round of selection on hVEGF followed by phage ELISA screeningand sequencing of VEGF-binding clones.

DNA Sequence Analysis

The high quality sequences from each library from the different targetselections were translated and aligned (Data not shown). The number ofsequences from each library subject to analysis is summarized in Table 4below.

TABLE 4 Number of Sequences Analyzed Library Total Sequences LCA-V2b 51LCH-V3 79 LCA-H2 97 LCH-H2 50 LCA-gD 112 LCH-gD 120 LCA-pL 60 LCH-pL 65HCA-V2 100 HCH-V2 96 HCA-H2 81 HCH-H2 96 HCA-gD 105 HCH-gD 105 HCA-pl102 HCH-pl 99The Wt/Mut ratios were calculated at each varied position (FIG. 35 andFIG. 36) thus allowing calculation of the F_(wt/mut) values as listed(FIG. 35 and FIG. 36) which are corrected for display by division of theratios from target selection by those from the display selection asdescribed (Vajdos et al., 2002). A F_(wt/mut) value greater than 1indicates that Wt is preferred at this position and F_(wt/mut) smallerthan 1 indicates the mutation is preferred. F_(wt/mut)>5 indicate itsimportant role in antigen binding. The importance of each scanned CDRresidue is illustrated in FIGS. 37A-37D. The result demonstrates thatresidues from both heavy chain and light chain contribute energeticallyto the binding of both antigen (Her2 and hVEGF) binding. The impact ofbH1 light chain and heavy chain residues on Her2 binding was compared tothat of its parent antibody hu4D5 (Kelley and O'Connell, 1993) (FIG.38).

FIGS. 39A1-39A3 and FIGS. 39B1-39B3 show shotgun alanine- and homologscanning of bH1 Fab for binding to VEGF and HER2. The effects ofmutation of alanine (ml), or additional mutations (m2, m3; due tolimitations of shotgun-alanine codons), or to a homologous amino acid(m4) are calculated as the ratio of occurrence of wild-type and mutants(wt/mut) among the clones binding to human VEGF (FIGS. 39A1-39A3) orHER2 (FIGS. 39B1-39B3). In cases where only the wild-type residueappeared, the ratios are shown as larger than “>” the wild-type count.The identity of the amino acid substitutions (m1-m4) is shown assuperscripts on the F values. When the wild-type residue is alanine, itwas substituted by glycine (ml). The “*” indicates the extent of the bH1residues that are buried upon VEGF or HER2 complex formation (*25-49% ofaccessible area buried, **50-75% of accessible area buried, ***greaterthan or equal to 75% of accessible area buried).

The residues that contribute significantly to the energetic interactionsmake up the functional paratopes, which constitute a subset of thestructural binding sites. In contrast to the extensive overlap betweenthe sites of antigen contact the two functional paratopes show limitedoverlap (FIGS. 32 and 40). In particular, based on shotgun scanningmutagenesis, the ΔΔG values (y-axis, kcal/mol) are plotted for eachmutation to alanine (black bar) or a homologous amino acid (white bar)for VEGF (FIG. 40A) or HER2 (FIG. 40B) binding. The “t” represents alower limit, as mutations were not observed at this position. The “*”indicates the extent of the bH1 residue surface area buried upon VEGF orHER2 complex formation. (*25-49% buried, **50-75%, ***>75%). The VEGFbinding interaction is mediated primarily by the LC CDRs with Tyr32 ofCDR-L1 and His91 of CDR-L3 as the core hot spot (ΔΔG_(wt/ala)>1.5kcal/mol). HER2 binding is mainly contributed by HC CDRs. FIG. 32 showscrystal structures where the bH1 and the Herceptin® antibody residuesare shaded on the Fab surface based on their functional importance (darkshading and white lettering, ΔΔG≧1.5 kcal/mol; intermediate shading andblack lettering, 1≧ΔΔG<1.5 kcal/mol; light shading and black lettering,0.5≧ΔΔG<1 kcal/mol of alanine mutation). The black dotted line outlinesthe contact area as in FIG. 28. The white dotted line depicts the divideof light and heavy chain.

For VEGF binding and HER2 binding, the functional paratope residues aredistributed across HC and LC signifying the synergy of the two chains.Trp95 of CDR-H3 is the only common hot spot residue for the twointeractions (ΔΔG_(wt/ala)>1.5 kcal/mol). As noted above, the VEGFbinding interaction is mediated primarily by the LC CDRs while HER2binding is dominated by HC CDRs. Compared to the Herceptin® antibody,bH1 with weaker HER2 binding affinity (300 fold) maintains the same corehot spot residues for HER2 binding (Arg50, Trp95, and Tyr100a) while theimportance of peripheral residues is redistributed (FIG. 32). Overall,most of the important side chains in heavy chain contributing hu4D5/Her2binding are still important for bH1/Her2 binding (ΔΔG>1.5 kcal/mol).There are some changes. Light chain residues have more shuffling incontributions—some residues became less important and some moreimportant. Overall, the functional sites are part of the structuralinterface from the crystal structure of the bH1-VEGF and bH1-Her2complexes.

In short, the interaction of bH1 with the two structurally unrelatedlarge proteins is characterized by the engagement of a distinct set ofbH1 residues in the energetic interaction with each antigen. While mostof the two extensively overlapping binding sites for the two differentantigens exhibit a single conformation, the flexibility of one CDR loop(L1) facilitates the accommodation of both HER2 and VEGF. The mechanismis reminiscent of the molecular versatility observed in multi-specificantibodies binding unrelated small haptens or peptides. Previous studiesdescribe multi-specificity mediated either by differential positioningof the small ligands at spatially distinct regions of a single antibodyconformation (Sethi et al., Immunity 24:429, 2006) or by multiplepre-existing conformations of the antigen binding site (James et al.,Science 299:1362, 2003). The versatility of antibody molecules inantigen recognition is further highlighted by how limited LC mutationscan give rise to antibodies that bind two unrelated protein antigens.

bH1 Affinity Maturation

In an attempt to investigate whether the VEGF-binding affinity of bH1could be increased by optimization of the light chain sequence beforethe structural and functional results became available, a library wasconstructed where the CDR residues at highly solvent-accessiblepositions based on the crystal structure of h4D5⁴²Fab (Eigenbrot et al.,2001), which is assumed to closely resemble bH1 Fab, were diversified.Targeted residues were allowed to vary as either wild type or a fewhomologous residues (FIG. 34). The library was constructed as describedin section “Construction of shotgun scanning libraries.” Asolution-based selection method was used to select for higher affinityVEGF-binders as described. Two rounds of solution-based selection wereperformed. The stringency was increased in each round of selection bydecreasing the concentration of biotinylated VEGF from 50 nM in thefirst round to 20 nM in the second round. 138 clones were sequenced fromthe last round of selection. Most clones were found to be unique. Ahigh-throughput ELISA assay with immobilized VEGF (8-109), anti-gDantibody, and Her2-ECD was used to identify clones that bound to VEGF,Her2-ECD, and anti-gD mIgG but not to BSA. The VEGF-ELISA bindingsignals were normalized by the anti-gD ELISA signals to estimate therelative affinity of the VEGF binding clones. Clones with highVEGF/anti-gD ratios were selected for further characterization. Theaffinity of the selected clones for VEGF and Her2 was estimated bycompetition ELISA as phage-displayed Fabs as previously described. ThebH1 variants show improved VEGF binding-affinity compared to the parentbH1 clone. Interestingly, some clones have slightly improved IC₅₀ valuesfor Her2 binding even though that affinity-based selection for Her2 wasnot performed. This shows that it is possible to affinity mature the bH1clone for VEGF binding without affecting Her2 binding abilitysignificantly. There are some VEGF-affinity improved clones that showedreduced Her2 binding affinity compared to the parent bH1 clone. Thisresult indicates that the light chain actively contributes to thebinding ability of bH1 to Her2 despite the fact that heavy chain is themain contributor to the binding energy based on the bH1-Her2 complexstructure and shotgun alanine scanning analysis. The sequences and IC₅₀values of the characterized clones are summarized in FIG. 41. Thefinding that most sequences were unique suggests that the light chainsequence of these variants is not yet fully optimized for VEGF bindingand that it is possible to further affinity-improve bH1 clone byadditional rounds of selection.

As shown in Table 5, significant affinity improvement of a single Fabfor two antigens is achievable and generally applicable. For instance,the K_(D) for human VEGF was increased from 250 (bH1; IgG) to 41(bH1-81; IgG) or 16 nM (bH1-44; IgG) and the K_(D) for HER2 wasincreased from 21 (bH1; IgG) to 7 (bH1-81; IgG) or 1 nM (bH1-44; IgG).

The affinity was improved by introducing mutations in the HC and LC CDRsof bH1. The positions were selected based on the information about thefunctional paratopes for VEGF and HER2 described herein. The bH1variants were isolated in two steps by selection and screening of phagedisplay libraries as described herein. The improved clone bh1-81 wasisolated by affinity-based selections of the described light chainhomolog shotgun scan library. In the second step, the highest affinityclone (bH1-44) was isolated from a library by randomizing residues ofbH1-81. In particular, oligonucleotides were designed that randomizedsites in the HC and the LC of bH1-81 (Table 5) to encode ˜50% wild-typeand 50% of all other 19 amino acids at each position (Gallop et al.,Journal of Medicinal Chimistry 37:1233, 1994).

The K_(D)5 of bH1 affinity-improved variants (Table 5) were measured forFab fragments and IgG antibodies. Fab fragments and IgG antibodies wereexpressed in E. Coli and 293 cells respectively, and purified asdescribed herein. Surface plasmon resonance (SPR) measurements withBIAcore3000 were used to determine the binding affinities of Fabfragments and IgG antibodies as described in Lee et al. (J. Mol. Biol.340:1073, 2004). To study the affinity of the antibody as monovalent Fabfragments, the antigens (hVEGF₁₀₉, murine VEGF₁₀₂, and HER2 ECD) wereimmobilized at low density on a BIAcore CM5 chip. Serial dilutions ofFab fragments were contacted with the immobilized antigens and thebinding responses measured by SPR. A 1:1 Langmuir binding model was usedto calculate the k_(on), k_(off), and K_(D). To determine the K_(D) ofthe IgG antibodies, the IgG was captured on a BIAcore CM5 chip byimmobilized anti-Fc antibody and exposed to serial dilutions ofhVEGF₁₀₉, murine VEGF₁₀₂, and HER2-ECD. For HER2 a simple 1;1 Langmuirbinding model was used to determine the K_(D), while VEGF required abivalent analyte model. All the experiments were performed at 30° C.

Table 5 shows the randomized positions in bold and summarizes the CDRsequences (SEQ ID NOS:1-9 and 39-41) of bH1, bH1-81, and bH1-44 andtheir affinities (as determined by surface plasmon resonance).

TABLE 5 Variants of bH1 with improved dual affinity Light Chain CDR-L1CDR-L2 CDR-L3 Antibody 28 29 30 30a 30b 30c 30d 31 32 50 51 52 53 54 5591 92 93 bH1 D I P R S I S G Y W G S Y L Y H Y T bH1-81 N A K T F SbH-44 N A K T F S Light Chain Kd (nM) Heavy Chain CDR-L3 hVEGF mVEGFHER2 CDR-H1 CDR-H2 Antibody 94 95 96 Fab IgG Fab IgG Fab IgG 28 29 30 3132 33 50 51 52 bH1 T P P 300 250 >1000 >1000 26 21 N I K D T Y R I YbH1-81 S 58 41 ND 150 6 7 bH-44 S 9 16 33 36 0.7 1 S G Heavy ChainCDR-H2 CDR-H3 Antibody 52a 53 54 55 56 57 58 95 96 97 98 99 100 100a100b 101 102 bH1 P T N G Y T R W G G D G F Y A M D bH-81 bH-44 S E V VND = not determined.The monovalent affinity of the antibodies for human VEGF₁₀₉, murineVEGF₁₀₂, and HER2 ECD was measured by BIAcore. Table 5 showsrepresentative dissociation constants (K_(d)) for each bindinginteraction. The receptor-binding fragment of VEGF (VEGF₁₀₉) was used inthe BIAcore experiment because the bH1 variants bind the full-lengthprotein (VEGF₁₆₅) and VEGF₁₀₉ with similar affinity in solutioncompetition experiments (data not shown). Different assay formats andevaluation models were used to calculate the K_(d) for Fab fragments/IgGantibodies as described herein. The different assay/evaluation formatsyielded consistent dissociation constants for the individualinteractions.

Example 5 Analysis of IgG Activity in Cell Assays

To determine whether the bH1 and 3-1 antibodies could inhibit hVEGF₁₆₅induced proliferation of human umbilical vein endothelial (HUVEC) cells,they were tested in a proliferation assay. Human umbilical veinendothelial cells (HUVEC) (Cambrex, East Rutherford, N.J.) were grownand assayed as described (Fuh et al., J. Biol. Chem. 273:11197, 1998).Approximately 4000 HUVECs were plated in each well of the 96-well cellculture plate and incubated in Dulbecco's modified Eagle's/F-12 medium(1:1) supplemented with 1.0% (v/v) fetal bovine serum (assay medium) for18 hours. Fresh assay medium with fixed amounts of VEGF (0.2 nM finalconcentration), which was first titrated as a level of VEGF that canstimulate submaximal DNA synthesis, and increasing concentrations ofanti-VEGF antibodies (e.g., bH1) were then added to the cells. Afterincubation at 37° C. for 18 hours, cells were pulsed with 0.5 μCi/wellof [³H]Thymidine for 24 hours and harvested for counting with TopCountMicroplate Scintillation counter. The results demonstrate that both 3-1and bH1 can inhibit VEGF-induced growth of HUVEC cells by preventinghVEGF induced signaling and subsequent proliferation. The Avastin®antibody (anti-VEGF) was used as a positive control and the Herceptin®antibody as a negative control (FIG. 42).

To study binding of bi-specific anti-Her2/VEGF antibodies to Her2expressed on mammalian cells, the binding of bH1 and bH3 antibodies toNR6 fibroblast cells overexpressing Her2 (NR6-Her2) was studied by FlowCytometry. One million NR6-Her2 cells were incubated with 100 μg/ml Faband IgG for 1 hour, followed by incubation with an Alexa488-conjugatedmurine anti-human IgG antibody for 1 hour. As negative controls, Fab andIgG binding to non-expressing NR6 cells was studied. As demonstrated inFIG. 43, bH1 and bH3 bind specifically to Her2 on NR6 cells as Fab andas IgG.

FIG. 44 shows the results of competitive binding experiments for bH1 toVEGF or HER2. bH1 inhibited VEGF induced proliferation of humanumbilical vein endothelial cells (HUVEC) with an IC₅₀ of 200 nM, whichis consistent with its affinity of 300 nM, and the proliferation ofHER2-expressing breast cancer cell line BT474 after 5-day incubation,albeit with lower efficiency than the Herceptin® antibody due to itsreduced affinity (FIG. 45). The Herceptin® IgG antibody and bevacizumab(anti-VEGF) served as controls. As shown in FIG. 45, bH1-81 and bH1-44antibodies inhibit VEGF-induced proliferation of HUVEC cells and growthof BT474 cells to a greater extent than bH1. The increased potencies ofthe bH1 variants correlate with their relative affinities. The highestaffinity variant, bH1-44, inhibits growth of HUVEC and BT474 cells witha potency similar to bevacizumab or Herceptin® antibody, respectively.

To carry out these experiments, VEGF-stimulated HUVECs were treated withincreasing concentrations of human IgG and the proliferation inhibitionafter 2-days of incubation was measured as described in Liang et al. (J.Biol. Chem. 281:951, 2006). Breast cancer cells BT474 were cultured inRPMI media supplemented with 10% FBS. For the assays, 10⁴ cells wereplated per well in a 96-well plate and incubated overnight (18 hours) at37° C. Increasing concentrations of human IgG were added to the cells.The cells were then incubated at 37° C. for five days, followed byaddition of 10% AlamarBlue (Biosource International, Camarillo, Calif.)according to the manufacturer's instructions. The antibody-dependentinhibition of proliferation of the HER2 expressing cells was determinedby measuring the fluorescent signal after 6 hours.

Example 6 Analysis of Binding Specificity

The binding specificity of the antibodies derived from the LC librarywas determined. IgGs binding to various immobilized purified proteins orcell lysates including the cognate antigens was assayed by ELISA. Theantigens were immobilized and incubated with hIgG at a concentration of15 μg/mL for an hour. Bound IgG were detected spectrophotometrically(optical density at 450 nm; y-axis; FIG. 46). The proteins included inthe assay were (left to right in FIG. 46): vascular endothelial growthfactor A (VEGF), murine vascular endothelial growth factor (murineVEGF), vascular endothelial growth factor C, (hVEGF-C), vascularendothelial growth factor D, (hVEGF-D), HER2 extracellular domain (HER2ECD), epidermal growth factor receptor extracellular domain (hEGFR),ErbB3/HER3 extracellular domain (HER3 ECD), human death receptor 5(hDR5), bovine serum albumin (BSA), Casein, Fetal Bovine Serum (FBS),Neutravidin, 5% milk, mouse fibroblast cell lysate, and mouse fibroblastcell lysate spiked with hVEGF-A or HER2 ECD. In FIG. 46, error barsrepresent the standard error means (SEM) of duplicates. The antibodiesbH3, 3-1, bD1, bD2, 4-1, and 4-5 were not tested for binding to murineVEGF, HER3 ECD, Neutravidin, 5% milk, cell lysate spiked with hVEGF-A,and cell lysate spiked with HER2 ECD.

The ability of various antibodies (Avastin® antibody, Herceptin®antibody, bH1, bH3, bH4, bH1-81, and bH1-44) to block VEGF binding toVEGF receptors was also determined (FIG. 47). Biotinylated human VEGF₁₆₅(FIG. 47A) or murine VEGF₁₆₄ (FIG. 47B) were equilibrated withincreasing concentrations of IgG (x-axis). Unbound VEGF was captured onimmobilized human VEGFR2-ECD Fc fusion protein and detectedspectrophotometrically (optical density at 450 nm, y-axis) Similarinhibition was also observed with VEGFR1. The anti-VEGF antibodies blockVEGF binding to VEGF receptors.

The antigens VEGF and HER2 were shown to compete for binding to bH1-44bispecific IgG antibody in solution (FIG. 48). Human bH1-44 IgG antibodyat a concentration of 0.1 nM was incubated with 0.1 nM biotinylatedhuman VEGF₁₆₅ in the presence of increasing concentrations of HER2 ECD.bH1-44 was captured by immobilized anti-human Fc and bH1-44-boundbiotin-VEGF detected with streptavidin-HRP. HER2 ECD bound to capturedbH1-44 was detected using a murine anti-HER2 antibody binding anon-overlapping epitope on HER2 followed by an HRP-conjugated goatanti-mouse IgG (FIG. 48A). Human bH1-44 IgG at a concentration of 0.2 nMwas incubated with 0.6 nM biotinylated HER2 in the presence ofincreasing concentrations of human VEGF₁₆₅. bH1-44 was captured byimmobilized anti-human Fc and bH1-44-bound biotin-HER2 detected withstreptavidin-HRP (FIG. 48B).

The specific binding of bH1 and bH1-44 to cells as also detected byusing FACS (Fluorescence Activated Cell Sorting; FIG. 49). Thebispecific antibodies (bH1 and bH1-44) bind to HER2 expressing mousefibroblast (NR6) cells (FIG. 49B) but not to HER2 negative NR6 cells(FIG. 49A). 0.5-1 million cells were incubated with 15 μg/mL hIgG on icefor an hour. Primary antibodies bound to cells were detected using asecondary fluorescent PE conjugated goat-anti-human IgG. The cells wereanalyzed using a FACS Calibur flow cytometer. bH1 and bH1-44 do notcross react with the rat ortholog of HER2, as no binding was detected tomouse fibroblast cells transfected with rat neu (rat ortholog of HER2).

To further characterize the specificity of the bH1 antibody variantsbH1-81 and bH1-44, immunoprecipitation experiments were conducted andthe bH1 antibody variants were shown to specifically immunoprecipitateVEGF or HER2 from mouse fibroblast (NR6) lysates, but not other proteins(FIG. 50). NR6 cells were non-specifically biotinylated, lysed, and cellmembrane proteins detergent solubilized. Cell lysates corresponding to5-10 million cells/mL of NR6 cells, NR6 cells spiked with 0.1 μg/mLbiotinylated VEGF₁₆₅, or HER2 over expressing NR6 cells were incubatedwith 15 μg/mL antibody. The antibody was captured using proteinA-coatedsepharose beads and bound proteins eluted. The eluted proteins wereseparated by SDS-PAGE. Cell lysates corresponding to approximately25-50,000 cells and immunopreciptate from approximately 0.12-0.25million cells were loaded onto the gel. Captured biotinylated proteinswere detected by Western blotting using streptavidin-HRP.

Example 7 Analysis of IgG Activity in In Vivo Assays

To assess whether the dual activity of these antibodies in vitrotranslates to a corresponding activity in vivo, we employed mousexenograft tumor models known to be responsive to treatment by anti-VEGFantibody (Colo205, a colorectal cancer cell line) or Herceptin® antibody(BT474M1, breast cancer cell line). In particular, Colo205 xenograftswere used in nu/nu mice and BT474M1 xenografts were used in beige nudeXID mice. All animal studies were in accordance with the guidelines ofthe American Association for Accreditation of Laboratory Animal Care andthe Genentech Institutional Animal Care and Use Committee.

In particular, the BT474M1 (in-house) and Colo205 (ATCC, Manassas, Va.)cells were cultured in RPMI media/10% fetal bovine serum. 5×10⁶ BT474M1cells suspended in Hank's Buffered Salt Solution (HBSS) and matrigel(1:1) mixture were injected into the mammary fat pad of Harlan beigenude XID mice (Indianapolis, Ind.) implanted with an estradiol pelletsubcutaneously. For Colo205 xenografts, 5×10⁶ Colo205 cells in HBSS weresubcutaneously injected into Charles River nu/nu mice (Hollister,Calif.). When the mean tumor size reached ˜200 mm³, the mice wererandomly grouped into 7 groups of 8 mice (BT474M1) or 10 mice (Colo205).Antibodies were administered intraperitoneally once a week. The tumorsizes were measured twice a week. Volumes were calculated as V=0.5ab² (ais the longest dimension of the tumor and b perpendicular to a). Thestatistical evaluation used one-way analysis followed by two-tailedstudent t tests. Adjustment of the alpha level due to multiplecomparisons (Bonferroni) did not alter the significance of ourconclusions. Partial responses (PR) were defined as a response of 50-99%reduction in tumor volume compared to V0. Serum samples were collected 7days after the first and third treatment. The concentration of humanantibody was determined using ELISA. Donkey anti-human IgG Fc wasimmobilized on an immuno plate. Dilutions of serum and an antibodystandard were incubated on the plate for 2 hours. Bound antibody wasdetected by Horseradish Peroxidase conjugated goat anti-human IgG Fcfollowed by TMB Substrate/1M Phosphoric Acid. The plates were read at450/620 nm. Sample concentrations were determined using a 4-parameteralgorithm.

The bH1-44 treated groups were compared with groups treated withanti-VEGF (B20-4.1) (Liang et al., J. Biol. Chem. 281:951, 2006),Herceptin® antibody, or the combination (Herceptin® antibody+anti-VEGF)to further establish that bH1-44 antibody was capable of inhibiting VEGFand HER2 mediated tumor growth. In all groups, antibody was present inserum from Colo205 xenografts at high levels (estimated by ELISA) 7 daysafter the start of treatment, indicating normal pharmacokinetics (Table6).

TABLE 6 Antibody serum levels Antibody Concentration (μg/ml) Group MeanSD Control IgG 10 mg/kg 65 14 Herceptin ® 10 mg/kg 83 47 Anti-VEGF 10mg/kg 20 8 Anti-VEGF + Herceptin ® 41 25 10 + 10 mg/kg bH1-44 10 mg/kg30 12 bH1-44 20 mg/kg 37 9 For each group, n = 5; SD = StandardDeviation

bH1-44 dosed weekly at 10 mg/kg inhibited Colo205 tumor growth comparedto control antibody (p<0.0001, n=10), with similar efficacy as anti-VEGF(10 mg/kg/week), while Herceptin® antibody had no effect on Colo205growth (p=0.12, n=10). As expected, the combination treatment showedsimilar efficacy as anti-VEGF alone. bH1-44 antibody administered at 10and 20 mg/kg/week yielded dose-dependent responses. In the BT474M1model, significant tumor growth inhibition was observed in the group ofmice treated with bH1-44 antibody (10 mg/kg/week, p=0.0005, n=8 and 20mg/kg/week, p=0.0001, n=7). Like the groups dosed with Herceptin®antibody or Herceptin®/antiVEGF combination, more than half of thetumors treated with bH1-44 antibody showed regression of more than 50%from the initial volume (i.e., partial response, FIG. 51). Anti-VEGFalone, on the other hand, only exhibited modest growth inhibitoryeffects on BT474M1 compared to control (p=0.06, n=7) and exhibited nopartial response. The bispecific bH1-44 antibody was thus shown toinhibit two distinct mechanisms important for tumor growth in vivo.

The above results indicate the potential of the affinity-improvedvariants of bH1 antibody (e.g., bH1-44 and bH1-81) to inhibit twomechanisms that are important for tumor growth in vivo.

Example 8 Characterization of VEGF and HER2 Binding Interfaces with bH1and bH1-44

To further compare the structural characteristics of bH1 and bH1-44, theVEGF and HER2 binding interfaces with these antibodies were identified.The structural contacts listed in Table 7 were identified based on thecrystal structure coordinates 3BDY (bH1/VEGF) and 3BE1 (bH1/HER2). Thebinding interface was calculated using the program XSAE. This programdefined the interface as polar, hydrophobic, and mixed. Table 7 liststhe bH1 residues with >25% of the total surface area buried upon HER2 orVEGF binding. Table 7 also lists the VEGF and HER2 residues within 4.5 Åof the bH1 residues. The surface area of each residue that is buriedupon complex formation was calculated using IMOL based on thecoordinates of the crystal structures 3BDY, 3BE1, and 1N8Z (PDB). Thepolar and hydrophobic interface areas reported in Table 11 reflect thepolar interface area and half of the mixed. The hydrophobic interfacearea reported consists of the hydrophobic areas and half of the mixed.

The crystal structure and alanine scanning showed that bH1 retains thesame binding epitope on HER2 as the Herceptin® antibody (Bostrom et al.,2009). The crystal structure of Herceptin® Fab in complex with HER2superimposes well onto the bH1/HER2 complex (r.m.s.d of 0.8 Å) (Bostromet al., 2009; Cho et al., 2003). Further, the Herceptin® antibodyresidues that contribute more than 10% of the total binding energy basedon alanine scanning mutagenesis are conserved, and many of them are alsopart of the binding hotspots of bH1 and bH1-44 (Bostrom et al., 2009;Kelley and O'Connell, 1993) (Table 14, FIG. 62). The interfaces betweenbH1/VEGF and bH1/HER2 bury 1506 Å² and 1579 Å², respectively, and aremainly hydrophobic (60% and 63%, respectively). The Herceptin®/HER2binding interface has similar size and composition as the bH1/HER2interface (1524 Å², 60% hydrophobic, Table 11), and is alsocharacterized by high shape complementarity (Table 8) (Bostrom et al.,2009).

TABLE 7 List of structural contacts of the complex of bH1 Fab/HER2 ECDand bH1/Fab/VEGF₁₀₉. The table lists residues with >25% of the totalsurface area buried upon HER2 and VEGF binding. The VEGF and HER2residues within 4.5 Å of the bH1 residues are listed. The surface areaof each residue that is buried upon complex formation was calculatedusing IMOL based on the coordinates of the crystal structures 3BDY,3BE1, and 1N8Z (PDB). Area buried Area buried bH1 residue by HER2 (%)HER2 residues contacting bH1 by VEGF (%) VEGF residues contacting bH1Heavy Y33 48 E558 F573 87 H86 Chain R50 97 E558 D560 F573 35 H86 Y52 30H86 Y56 42 P557 E558 R58 50 E558 Q561 W95 100 P572 F573 61 H86 Q87 G9993 D570 P579 K593 75 K48 I83 Q89 Y100a 80 D570 P571 P572 F573 88 I83 K84P85 H86 Q87 G88 Q89 Light S28 59 I91 G92 E93 Chain I29 77 R82 H90 I91G92 S30 69 H90 I91 G31 85 G88 Q89 H90 I91 Y32 97 D570 P571 A600 C601Q602 89 Q89 H90 W50 62 K593 P603 59 F17 M81 Q89 Y53 44 P603 C604 P605 74F17 M18 I91 H91 90 P571 P572 81 G88 Q89 Y92 55 K569 P571 P572 76 Y45 K84G88 Q89 H90 T93 61 K84 Q87 G88 T94 68 D560 P572 55 H86 Q87

The shape complementarity (represented as Sc in Table 8) between theantibody and the antigen was determined as described (Lawrence et al.,1993). The high shape complementarity in the bH1/VEGF and bH1/HER2complexes, similar to the complementarity between the Herceptin®antibody and HER2, are in the range of reported antibody-antigencomplexes (Sc 0.64-0.68; Lawrence et al., 1993). Superposition of HER2with bH1 in its VEGF-bound conformation or VEGF with bH1 in itsHER2-bound form reveals little shape complementarity observed whenjuxtaposing an antibody with an unrelated antigen. (Sc 0.35; Lawrence etal., 1993). The results demonstrate the extent to which bH1 rearrangesto accommodate the two different antigens.

TABLE 8 Different surface conformations of bH1 for binding HER2 andVEGF. Shape complementarity in antibody/antigen complexes AntibodyAntigen Sc* Herceptin HER2 0.75 bH1 HER2 0.72 bH1 VEGF 0.68 bH1(VEGF-bound conformation) HER2 0.40 bH1 (HER2-bound conformation) VEGF0.44 *Sc = Median Shape Complementarity Statistic

The affinity of bH1 was improved by selecting the high affinity variantbH1-44 from phage-displayed antibody libraries of bH1. Shotgun alaninescanning mutagensis demonstrated that bH1-44 conserved the hotspot forantigen binding of bH1 (Tables 9A-B, 10, and 14). Shotgun alaninescanning mutagenis of bH1-44 was performed using the techniquesdescribed above for the shotgun alanine scanning mutagenesis of bH1.

In Tables 9A-B the effects of mutation to alanine (ml), or additionalmutations (m2, m3; due to limitations of shotgun codons), or to ahomologous amino acid (m4) are calculated as the ratio of occurrence ofwild type (wt) or wt/mut for VEGF (Table 9A) or HER2 (Table 9B) bindingclones. When the wt was alanine, it was substituted by glycine (ml). Thewt/mut ratios are corrected for protein folding/expression effects bydivision with wt/mut ratios from display selection to obtain the Fvalues. Display selection was performed independently by selectingclones binding to protein L, which binds a non-linear epitope of theantibody light chain. As only the Fab heavy chain is fused to the phagecoat protein (p3), protein L binding indicates proper folding andassociation of light chain and heavy chain.

In Table 10, the antibody residues of bH1 and bH1-44 that contact VEGFand/or HER2 in the crystal structure are listed. The energetic hotspotsfor binding are defined by the antibody residues that result inΔΔG_(wt/ala) greater than approximately 10% of the total binding energyof the interaction.

The data in Table 11 indicate that the polarity and size of the bindinginterfaces are similar between bH1/VEGF, bH1/HER2, and theHerceptin®/HER2 complex. The polarity of each interface was analyzedusing XSAE. All the numbers depicted in Table 11 represent the area inÅ², unless otherwise indicated.

TABLE 9A Shotgun alanine- and homolog-scanning of bH1-44 Fab for bindingto VEGF. Antigen selection (VEGF) Display Selection (Protein L) Fwt/mutvalues ΔΔG_(wt/mut) (kcal/mol) wt/m1 wt/m2 wt/m3 wt/m4 wt/m1 wt/m2 wt/m3wt/m4 Fwt/m1 Fwt/m2 Fwt/m3 Fwt/m4 ΔΔG_(wt/m1) ΔΔG_(wt/m2) ΔΔG_(wt/m3)ΔΔG_(wt/m4) CDR-L1 Q27 0.3 1.0 9.0 1.2 0.8 0.9 0.4 2.8 0.4 1.1 20.3 0.5−0.6 0.04 1.8 −0.5 N28 1.8 0.6 3.5 1.2 0.6 0.9 1.0 8.4 2.8 0.7 3.5 0.10.6 −0.2 0.7 −1.1 I29 39.0 39.0 3.9 36.0 0.8 0.7 0.4 0.8 46.8 59.8 8.842.7 2.3 2.4 1.3 2.2 A30 2.8 NA NA 1.7 0.4 NA NA 0.2 6.5 9.9 1.1 1.4K30a 3.0 2.7 9.0 1.6 1.4 3.1 1.1 0.2 2.1 0.9 7.8 8.7 0.4 −0.1 1.2 1.3T30b 7.0 NA NA 1.4 0.9 NA NA 0.5 7.7 2.7 1.2 0.6 I30c 16.0 5.3 0.5 0.92.3 2.1 0.7 1.0 7.1 2.6 0.7 0.9 1.2 0.6 −0.2 −0.1 S30d 15.7 NA NA 74.01.7 NA NA 0.5 9.1 150.4 1.3 3.0 G31 24.0 NA NA 74.0 2.6 NA NA 3.0 9.424.3 1.3 1.9 Y32 46.0 23.0 46.0 74.0 0.5 1.9 0.3 1.6 99.1 12.4 152.245.9 2.7 1.5 3.0 2.3 CDR-L2 W50 46.0 15.3 46.0 74.0 2.3 1.5 2.4 1.1 20.410.2 19.2 65.1 1.8 1.4 1.7 2.5 G51 24.0 NA NA 74.0 7.3 NA NA 7.5 3.3 9.80.7 1.4 S52 15.7 NA NA 36.0 4.1 NA NA 7.5 3.9 4.8 0.8 0.9 F53 22.0 44.014.7 5.7 1.9 2.4 1.4 0.4 11.6 18.1 10.8 13.5 1.5 1.7 1.4 1.5 CDR-L3 H917.3 44.0 44.0 73.0 0.0 0.1 0.3 2.6 150.3 880.0 154.0 27.9 3.0 4.0 3.02.0 Y92 48.0 48.0 48.0 13.8 3.8 6.3 1.0 2.8 12.6 7.6 46.7 5.0 1.5 1.22.3 1.0 S93 1.0 NA NA 1.7 3.0 NA NA 2.5 0.4 0.7 −0.6 −0.2 S94 15.7 NA NA3.4 1.0 NA NA 0.9 15.3 3.7 1.6 0.8 CDR-H1 S30 1.2 NA NA 1.7 1.2 NA NA1.3 1.0 1.3 0.0 0.2 G31 2.4 NA NA 5.6 1.2 NA NA 5.0 2.0 1.1 0.4 0.1 T320.5 NA NA 0.5 0.9 NA NA 0.6 0.5 0.9 −0.4 −0.1 Y33 10.2 1.0 2.2 12.5 1.42.0 0.8 2.3 7.1 0.5 2.6 5.5 1.2 −0.4 0.6 1.0 CDR-H2 R50 0.6 0.9 20.5 0.42.1 1.3 70.0 1.3 0.3 0.7 0.3 0.3 −0.7 −0.2 −0.7 −0.8 Y52 1.8 30.0 7.51.4 2.0 2.5 1.8 2.7 0.9 11.9 4.3 0.5 −0.1 1.5 0.9 −0.4 S53 1.4 NA NA 1.01.2 NA NA 1.1 1.2 0.9 0.1 0.0 E54 1.2 NA NA 0.7 0.4 NA NA 1.0 3.4 0.70.7 −0.3 Y56 9.2 6.5 6.9 0.7 1.6 2.3 1.3 1.2 5.8 2.8 5.3 0.6 1.0 0.6 1.0−0.3 R58 1.5 1.8 8.1 1.3 2.1 2.3 4.9 2.7 0.7 0.8 1.6 0.5 −0.2 −0.2 0.3−0.4 CDR-H3 W95 139.0 139.0 8.7 243.0 0.8 0.2 0.3 4.5 185.3 685.7 27.253.7 3.1 3.9 2.0 2.4 V96 0.5 NA NA 1.4 2.1 NA NA 1.5 0.2 1.0 −0.9 0.0G97 0.8 NA NA 2.9 0.9 NA NA 5.7 0.9 0.5 −0.1 −0.4 V98 2.3 NA NA 1.2 1.5NA NA 0.8 1.6 1.4 0.3 0.2 G99 2.1 NA NA 3.0 1.2 NA NA 1.8 1.7 1.7 0.30.3 F100 6.2 9.5 5.0 2.2 2.0 2.0 0.9 1.8 3.0 4.7 5.4 1.2 0.7 0.9 1.0 0.1Y100a 27.2 27.2 15.1 2.2 1.5 1.5 0.6 0.9 17.9 18.6 26.6 2.5 1.7 1.7 1.90.5 NA = Mutation not included.

TABLE 9B Shotgun alanine- and homolog-scanning of bH1-44 Fab for bindingto HER2. Antigen Selection (HER2) Display Selection (Protein L) Fwt/mutvalues ΔΔG_(wt/mut) (kcal/mol) wt/m1 wt/m2 wt/m3 wt/m4 wt/m1 wt/m2 wt/m3wt/m4 Fwt/m1 Fwt/m2 Fwt/m3 Fwt/m4 ΔΔG_(wt/m1) ΔΔG_(wt/m2) ΔΔG_(wt/m3)ΔΔG_(wt/m4) CDR-L1 Q27 0.9 1.0 0.6 1.4 0.8 0.9 0.4 2.8 1.2 1.0 1.3 0.50.1 0.0 0.1 −0.4 N28 1.3 0.9 1.2 1.1 0.6 0.9 1.0 8.4 2.0 1.0 1.2 0.1 0.40.0 0.1 −1.2 I29 1.5 1.9 0.8 0.8 0.8 0.7 0.4 0.8 1.8 3.0 1.7 1.0 0.3 0.60.3 0.0 A30 0.3 NA NA 1.1 0.4 NA NA 0.2 0.8 6.5 −0.2 1.1 K30a 1.5 2.61.8 1.1 1.4 3.1 1.1 0.2 1.1 0.8 1.6 6.0 0.0 -0.1 0.3 1.1 T30b 1.0 NA NA0.3 0.9 NA NA 0.5 1.1 0.6 0.1 −0.3 I30c 4.4 5.3 1.8 1.5 2.3 2.1 0.7 1.01.9 2.6 2.6 1.6 0.4 0.6 0.6 0.3 S30d 0.9 NA NA 1.0 1.7 NA NA 0.5 0.5 2.1−0.4 0.4 G31 2.0 NA NA 2.2 2.6 NA NA 3.0 0.8 0.7 −0.1 −0.2 Y32 0.0 1.00.02 1.9 0.5 1.9 0.3 1.6 0.1 0.5 0.1 1.2 −1.8 -0.4 -1.6 0.1 CDR-L2 W508.1 24.3 8.1 91.0 2.3 1.5 2.4 1.1 3.6 16.2 3.4 80.1 0.8 1.7 0.7 2.6 G518.4 NA NA 44.5 7.3 NA NA 7.5 1.2 5.9 0.1 1.1 S52 8.4 NA NA 6.6 4.1 NA NA7.5 2.1 0.9 0.4 −0.1 F53 3.1 9.8 2.0 0.4 1.9 2.4 1.4 0.4 1.6 4.0 1.5 1.00.3 0.8 0.2 0.0 CDR-L3 H91 1.7 58.0 58.0 5.5 0.05 0.1 0.3 2.6 34.01160.0 203.0 2.1 2.1 4.2 3.1 0.4 Y92 22.5 90.0 90.0 4.1 3.8 6.3 1.0 2.85.9 14.2 87.6 1.5 1.1 1.6 2.7 0.2 S93 1.5 NA NA 2.8 3.0 NA NA 2.5 0.51.1 −0.4 0.1 S94 30.3 NA NA 7.3 1.0 NA NA 0.9 29.7 8.1 2.0 1.2 CDR-H1S30 1.3 NA NA 1.0 1.2 NA NA 1.3 1.0 0.8 0.0 −0.1 G31 1.5 NA NA 3.3 1.2NA NA 5.0 1.3 0.7 0.1 −0.2 T32 0.6 NA NA 1.5 0.9 NA NA 0.6 0.7 2.6 −0.20.6 Y33 150.0 150.0 150.0 5.7 1.4 2.0 0.8 2.3 104.3 75.0 179.3 2.5 2.82.6 3.1 0.5 CDR-H2 R50 150.0 150.0 150.0 134.0 2.1 1.3 70.0 1.3 70.7113.6 2.1 100.5 2.5 2.8 0.5 2.7 Y52 1.0 1.5 0.9 1.2 2.0 2.5 1.8 2.7 0.50.6 0.5 0.4 −0.4 -0.3 -0.4 −0.5 S53 1.0 NA NA 1.1 1.2 NA NA 1.1 0.9 1.0−0.1 0.0 E54 1.2 NA NA 2.2 0.4 NA NA 1.0 3.4 2.2 0.7 0.5 Y56 49.0 147.0147.0 0.9 1.6 2.3 1.3 1.2 31.2 64.1 112.3 0.7 2.0 2.5 2.8 −0.2 R58 23.7142.0 142.0 66.0 2.1 2.3 4.9 2.7 11.2 61.4 28.8 24.8 1.4 2.4 2.0 1.9CDR-H3 W95 150.0 150.0 150.0 134.0 0.8 0.2 0.3 4.5 200.0 740.0 470.029.6 3.1 3.9 3.6 2.0 V96 0.9 NA NA 1.2 2.1 NA NA 1.5 0.4 0.8 −0.5 −0.1G97 1.8 NA NA 6.9 0.9 NA NA 5.7 2.0 1.2 0.4 0.1 V98 0.6 NA NA 1.5 1.5 NANA 0.8 0.4 1.8 −0.5 0.3 G99 6.5 NA NA 21.3 1.2 NA NA 1.8 5.2 12.0 1.01.5 F100 145.0 145.0 29.0 133.0 2.0 2.0 0.9 1.8 71.1 71.1 31.3 73.0 2.52.5 2.0 2.5 Y100a 149.0 149.0 149.0 6.9 1.5 1.5 0.6 0.9 98.0 101.9 262.77.8 2.7 2.7 3.3 1.2 NA = Mutation not included.

TABLE 10 The structural and functional paratopes for VEGF and HER2. VEGFonly HER2 only Shared contac LC-S30b HC-Y56 LC-Y32 LC-I30c HC-R58 LC-W50LC-S30d LC-Y53 LC-G31 LC-H91 LC-T93 LC-Y92 LC-T94 HC-Y33 HC-R50 HC-W95HC-G99 HC-Y100a Hotspot LC-I29 LC-T94 LC-W50 residues LC-S30b HC-Y33HC-W95 (bH1) LC-S30d HC-R50 LC-G31 HC-Y56 LC-Y32 HC-R58 LC-G51 HC-G99LC-H91 HC-F100 LC-Y92 HC-Y100a Hotspot LC-I29 HC-Y33 LC-H91 residuesLC-T30b HC-R50 LC-S94 (bH1-44) LC-S30d HC-Y56 HC-W95 LC-G31 HC-R58LC-Y32 HC-F100 LC-W50 HC-Y100a LC-F53 LC-Y92 HC-Y100a

TABLE 11 The polarity and size of the binding interfaces of bH1/VEGF,bH1/HER2, and Herceptin ®/HER2 complexes. bH1 Fab/VEGF binding interfacebH1 Fab/HER2 binding interface Herceptin Fab/HER2 binding interface bH1VEGF Combined Percent (%) bH1 HER2 Combined Percent (%) Herceptin HER2Combined Percent (%) Polar 331 295  607 40% 308 282  591 37% 307 308 614 40% Hydrophobic 438 462  900 60% 470 518  988 63% 441 469  910 60%Total 749 757 1506 779 800 1579 747 777 1524HER2/VEGF Dual Specific bH1-44 Antibody Maintains the HER2 BindingKinetics of the Herceptin® Antibody

Surface plasmon resonance was performed to study the binding kinetics ofbH1 and its Fab variants to immobilized VEGF or HER2 (Table 12). AnSPR-based assay was performed using a BIAcore 3000. VEGF₁₀₉ and HER2extracellular domains were immobilized on CM5 chips at a density thatallowed for an Rmax in the range of 50-150 RU. Serial dilutions of Fabsin PBS with 0.05% Tween20 were injected at 30 μl/min. The bindingresponses were corrected by subtracting responses from a blank flow celland by normalizing for buffer effects. A 1:1 Langmuir fitting model wasused to estimate the k_(a) (onrate) and k_(d) (offrate). The K_(D)values were determined from the ratios of k_(a) and k_(d).

The bH1 Fab/VEGF interaction is characterized by a relatively highon-rate (k_(on)=3.7×10⁴) and a fast off-rate (k_(off)=0.013), whichresults in a moderate K_(D) of 300 nM. The affinity of the bH1/HER2interaction (K_(D)=26 nM, k_(on)=9.6×10⁴, k_(off)=2.4×10⁻³) is 52-foldlower than the Herceptin®/HER2 interaction (K_(D)=0.5 nM,k_(on)=7.1×10⁵, k_(off)=3.5×10⁴) with a slower on-rate and fasteroff-rate. The affinity improved bH1 variants, bH1-81 and bH1-44,displayed improvements in both the on-rates and off-rates of the VEGFand HER2 interactions. The high affinity clone bH1-44 binds HER2 with anaffinity similar to Herceptin® (K_(D)=0.2 nM, Table 12).

Table 12 depicts the kinetic profiles of the bH1 variants and theHerceptin® antibody determined by surface plasmon resonance measurementusing BIAcore at 30° C. In these experiments, Fabs were bound toimmobilized VEGF or HER2, and the on-rate (k_(a)), off-rate (k_(d)), anddissociation constant (K_(D)) were determined using a 1:1 Langmuirbinding fitting model. The bH1-44 antibody has a similar kinetic profileand affinity for HER2 as the Herceptin® antibody. The two double mutants(bH1-44 I29A+Y32A and bH1-44 R50A+R58A) that lost binding to VEGF orHER2 retained the kinetic profile and affinity for the other antigen.

TABLE 12 Kinetic profiles of the bH1 variants and the Herceptin ®antibody. VEGF₁₀₉ HER2 ECD ka (1/Ms) kd (1/s) K_(D) (nM) ka (1/Ms) kd(1/s) K_(D) (nM) Herceptin ® Fab — — NB 7.1E+05 3.5E−04  0.5 +/− 0.06Herceptin ® (R50A) Fab — — NB 2.7E+04 2.0E−03 74 Herceptin ® (R58A) Fab— — NB 5.9E+04 7.3E−04 12 Herceptin ® — — NB — — NB (R50A + R58A) FabbH1 Fab 3.7E+04 0.013 300 +/− 87  9.6E+04 2.4E−03 26 +/− 28 bH1-811.2E+05 0.007 58 +/− 12 2.2E+05 1.4E−03   6 +/− 0.6 bH1-44 Fab 4.0E+050.001   3 +/− 0.3 3.7E+05 8.0E−05  0.2 +/− 0.07 bH1-44 (Y32A) Fab — —weak 6.2E+05 3.5E−05 0.1 bH1-44 (I29A + Y32A) Fab — — NB 4.2E+05 8.3E−05 0.2 +/− 0.07 bH1-44 (R50A + R58A) Fab 3.5E+05 0.001   3 +/− 0.7 — — NBNB = No binding detectable.Dual Specific Antibodies Interact with HER2 and VEGF with SimilarThermodynamic Properties

The enthalpy (ΔH) and entropy (ΔS) changes for the interactions betweenthe bH1 Fab variants and the two antigens, VEGF (the receptor bindingdomain of VEGF, VEGF₈₋₁₀₉) and HER2 extracellular domain (ECD) were alsodetermined (FIGS. 59A-59F, FIG. 60, Table 13), using isothermaltitration calorimetry (ITC).

Microcalorimetric measurements of the interactions between Fabs andhuman VEGF₁₀₉ and the extracellular domain of HER2 were performed on aVP-ITC titration calorimeter (Microcal Inc.) as described (Starovasniket al., 1999). Protein solutions were extensively dialyzed intophosphate-buffered saline. The antigen and Fabs were dialyzed in thesame vessel to minimize mixing heat effects due to differences in buffercomposition. Fabs at a concentration of 100-220 μM were titrated intoantigen solutions (HER2-ECD or VEGF₁₀₉) at a concentration of 10-22 μM.This concentration of antigen was required for precise enthalpymeasurements, but precludes determination of the K_(D) in cases wherethe binding affinity is high. Fifteen or twenty injections wereperformed to obtain a 2-fold excess of antibody. The heats of reactionwere determined, heats of Fab dilution were subtracted, and the ΔH wascalculated.

The dissociation constants (K_(D)) determined by surface plasmonresonance (Table 12) were used to calculate the binding free energy (ΔG)according to:

ΔG=RT ln(K _(D))

The entropy change upon association (ΔS) was calculated according to:

ΔS=(ΔH−ΔG)/T, where T is the temperature (K).

To determine the ΔCp, microcalorimetric measurements were performed asdescribed above at different temperatures ranging from 20 to 37° C. TheΔCp was determined by linear regression by plotting ΔH as a function ofthe temperature (FIG. 62).

The interactions of the dual specific antibody, bH1, with either of itstwo antigens, VEGF and HER2 were first characterized. The binding of bH1with VEGF and HER2 exhibited similar thermodynamic properties (Table13). Both interactions, measured at 30° C. in PBS at pH 7.4, areexothermic (ΔH=−2.4 and −2.4 kcal/mol for VEGF and HER2, respectively,Table 13, FIG. 60) with a highly favorable entropy change contributingto the binding energy (−TΔS=−6.6 and −7.9 kcal/mol for VEGF and HER2,respectively, Table 13, FIG. 60).

Table 13 depicts the ΔG (binding free energy), ΔS (entropy change), andΔH (enthalpy change) in kcal/mol. The affinities shown were measured inat least two independent experiments using kinetic analysis by BIAcoreat 30° C. The ΔH was measured using ITC and represents the average oftwo or three independent measurements followed by the standarddeviations. The ΔG and ΔS were calculated as described above.

The high affinity variants bH1-81 and bH1-44 displayed similarthermodynamic profiles as bH1. Their interactions with VEGF and HER2were also characterized by favorable enthalpy and entropy (Table 13,FIG. 60). For the VEGF interaction, the affinity improvement wasassociated with a significantly more favorable enthalpy change (ΔH=−7.1for bH1-44 versus −2.4 kcal/mol for bH1 at 30° C.) and a slightly lesspositive entropy change (−TΔS=−6.6 for bH1-44 versus −4.7 for bH1 at 30°C., Table 13, FIG. 60). The improved affinity for HER2 was alsoassociated with a more favorable enthalpy change (ΔH=−5.3 versus −2.4kcal/mol, 30° C., Table 13, FIG. 60).

TABLE 13 Antigen binding affinities and thermodynamics for the bH1variants and the Herceptin ® antibody. VEGF₁₀₉ HER2-ECD K_(D) K_(D) (nM)ΔG ΔH −TΔS (nM) ΔG ΔH −TΔS Herceptin ® — — — — 0.5 −12.9 +/− 0.06 −13.6+/− 0.2  −0.3 +/− 0.2 bH1 300 −9.0 +/− 0.2 −2.4 +/− 0.7 −6.6 +/− 0.7 26−10.5 +/− 0.4  −2.4 +/− 0.5 −7.9 +/− 0.6 bH1-81 58  −10 +/− 0.1 −6.2 +/−0.1 −3.8 +/− 0.2 6 −11.4 +/− 0.05 −3.8 −7.6 bH1-44 3 −11.8 +/− 0.07 −7.1+/− 0.3 −4.7 +/− 0.3 0.2 −13.5 +/− 0.3  −5.3 +/− 0.4 −8.1 +/− 0.5 bH1-44— — — — 0.2 −13.5 +/− 0.3  −6.4 +/− 0.5 −7.6 +/− 0.6 (LC-I29A/Y32A)bH1-44 4 −11.6 +/− 0.1  −7.7 −3.9 — — — — (HCR50A/R58A)bH1-44 and Herceptin® Interact with HER2 with Distinct Thermodynamics

In contrast to the dual specific antibodies, the HER2/Herceptin®interaction is characterized by a large favorable enthalpy change(ΔH=−13.6 kcal/mol) without any significant entropy change (−TΔS=−0.3kcal/mol, FIG. 60, Table 13) (Kelley et al., 1992). Although bH1-44interacts with HER2 with similar affinity as Herceptin®, the bindingfree energy is made up of a greater entropy component (−TΔS=−8.1kcal/mol, 30° C.) and a smaller enthalpy component (ΔH=−5.3 kcal/mol,30° C.). The distinct thermodynamic properties contrast the manysimilarities in HER2 binding characteristics between Herceptin® andbH1-44, which include affinity, kinetics, and many of the residues ofthe energetic hotspots. Although the hot spot residues of Herceptin®that contribute more than 10% of the total binding energy for HER2 aresimilar to those of bH1 and bH1-44, there are some clear differences.

Table 14 shows the bH1, bH1-44, and the Herceptin® antibody hotspots forHER2 binding determined by alanine scanning mutagenesis. The mutagenesiswas performed as described in Kelley et al., 1993. The numbers in Table14 represent the change in binding free energy (ΔΔG_(wt-mut)) when theresidue is mutated to alanine. The hotspot residues in Table 14 areshaded and are defined as ΔΔG greater than or equal to 10% of the totalbinding free energy (ΔG).

Residues LC-Thr94, HC-Tyr33, HC-Asp98 are conserved in sequence in bH1but have different functions in HER2 binding (Table 14, FIG. 61). Hence,the mutations in the antigen-binding site of Herceptin® that recruitedVEGF binding appear to have made some fundamental changes to theantigen-binding site that affect the interaction with HER2. The dualspecific antibodies accommodate the introduced mutations by utilizing adifferent HER2 recognition strategy that results in equally highaffinity for HER2 as Herceptin®. It is interesting to note that exceptfor LC-Ser94 of bH1-44 the mutations that improved the affinity for HER2more than 100-fold compared to bH1 are not parts of the binding hotspot,but appear to optimize the existing interactions.

Large Negative Heat Capacities in the Dual Specific Interactions

To further understand the common energetics driving the dual specificinteractions and how they are distinguished from that of themonospecific parent Herceptin®, a series of experiments were performedto study following three Fab/antigen interactions: bH1-44 with VEGF orHER2, and Herceptin® with HER2. The heat capacities of the dual specificinteractions was measured by determining the enthalpy of binding (ΔH) atmultiple temperatures ranging from 20° C. to 37° C. (ΔT=17° C., FIG. 62,Table 15). The heat capacity (ΔCp) is a function of ΔH and Temperature(T) and can be described by the equation:

ΔCp=δ(ΔH)/δT

ΔCp was estimated from the slope of the temperature dependence of ΔH bylinear regression (FIG. 62, Table 15). ΔCp of bH1-44 was determined to−400 cal/molK for the interaction with VEGF, and −440 cal/molK for theinteraction with HER2. The large negative heat capacities indicate theimportance of the hydrophobic effect as previously described (Kauzmann,1959), which is consistent with the hydrophobic nature of the structuralinterfaces in the two complexes (Table 11). The ΔCp for Herceptin®/HER2,which was previously determined to −370 cal/molK in a similartemperature interval (Kelley et al., 1992), is smaller than the ΔCp ofbH1-44/HER2, but still indicates the important role of the hydrophobiceffect in HER2 binding.

The total entropy change (ΔS) of binding free energy is a sum of entropychanges from three sources (Murphy et al., 1994): entropy changesassociated with desolvation of the binding surfaces (ΔS_(SOLV)), entropychanges from the loss of rotational and translational degree of freedom(ΔS_(RT)), and entropy changes due to the changes in configurational andconformational dynamics of the interacting molecules (ΔS_(CONF)).

ΔS _(TOT) =ΔS _(SOLV) +ΔS _(RT) +ΔS _(CONF)  (1)

Typically, only ΔS_(SOLV) is positive while ΔS_(RT) and ΔS_(CONF) areboth negative. The cratic entropy term, ΔS_(RT), for the association oftwo molecules can be estimated to −8 cal/Kmol as described (Murphy etal., 1994). ΔS_(SOLV) can be assumed to be dominated by the hydrophobiceffect due to the burial of apolar surface area and can be described asa function of ΔCp:

ΔS _(SOLV) =ΔCp ln(T/T*), T*=385 K  (2)

ΔS_(CONF) can thus be estimated as:

ΔS _(CONF) =ΔS _(TOT) −ΔS _(RT) −ΔS _(SOLV)  (3)

According to equation (3), ΔS_(SOLV) is estimated to 96 calmol⁻¹K⁻¹ forbH1-44/VEGF, 105 calmol⁻¹K⁻¹ for bH1-44/HER2, and 89 calmol⁻¹K⁻¹ forHerceptin®/HER2 (Table 15). This translates to ΔS_(CONF) of −72calmol⁻¹K⁻¹ for bH1-44/VEGF, −70 calmol⁻¹K⁻¹ for bH1-44/HER2, and −80calmol⁻¹K⁻¹ for Herceptin®/HER2 (Table 15).

To examine the overall structural stability of the dual specific Fabscompared to its parent Herceptin®, thermal denaturation experimentsusing differential scanning calorimetry (DSC) were performed. Thermaldenaturation experiments were performed on a differential scanningcalorimeter from Microcal Inc. Fabs were dialyzed against 10 mM sodiumacetate pH 5, 150 mM sodium chloride. The solutions were adjusted to aconcentration of 0.5 mg/ml and heated to 95° C. at a rate of 1° C./min.The melting profiles were baseline corrected and normalized. The meltingtemperature (T_(M)) was determined using the software supplied by themanufacturer. As expected, none of the Fabs displayed reversible thermaldenaturation profiles (Kelley et al., 1992) (data not shown). The T_(M)of the dual specific variants (77.2° C., 75.6° C., 74.3° C. for bH1,bH1-81, and bH1-44, respectively, Table 16) are slightly lower than thatof Herceptin® (82.5° C.), but high and within the range of what has beenreported for other therapeutic antibodies (Garber and Demarest, 2007).

The Binding Kinetics and Thermodynamics of bH1 Variants with HighAffinity for Only VEGF or HER2

Interestingly, the dual specific antibodies derive the majority of theirbinding energy from entirely distinct regions of the shared VEGF/HER2binding site. These data show that the VEGF or HER2 binding function ofthe dual specific antibodies can be selectively disrupted withoutaffecting the remaining binding specificity. Structural studiesindicated that the structural paratopes on bH1 for VEGF and HER2 overlapsignificantly, but shotgun alanine mutagenesis of bH1 and bH1-44demonstrated that the VEGF and HER2 interactions are mediated by twounique sets of CDR residues with little overlap (FIGS. 54 and 57, Tables9A, 9B, and 10). The shotgun alanine scanning of bH1 and bH1-44indicated that some CDR residues are exclusively important for bindingeither VEGF or HER2 (FIGS. 54 and 57, Tables 9A, 9B, and 10), includingLC-Ile29, LC-Tyr32, which are important for VEGF binding, and HC-Arg50,HC-Arg58 for HER2 binding (FIGS. 54 and 57, Tables 9 and 10). To confirmthe unique importance of the side chains of these residues in eachinteraction, each residue was mutated to alanine in the bH1-44(LC-Ile29, LC-Tyr32, HC-Arg50, HC-Arg58) or the Herceptin® (HC-Arg50,HC-Arg58) scaffolds, individually or in combination, and expressed themutants as Fabs and IgGs.

Vectors that encoded bH1-44 or Herceptin® Fabs fused to the N-terminusof geneIII via the heavy chain was used as the templates for Kunkelmutagenesis (Kunkel et al., 1987). Oligonucleotides were designed tointroduce the desired alanine mutations at selected positions. The Fabalanine mutants were expressed as phage, and the binding verified bycompetition ELISA (FIG. 58). The heavy chain and the light chainvariable domains were then cloned into Fab and IgG expression vectors,and Fabs and IgGs expressed and purified as described (Bostrom et al.,2009). SDS-PAGE verified the correct protein size (FIG. 65). Sizeexclusion chromatography showed aggregation levels of less than 5%.

Binding to the two antigens was examined by competition ELISA and/orBIAcore. All single alanine mutations in the bH1-44 scaffold impairedbinding to varying degrees (data not shown). The most striking singlemutation was LC-Y32A, which significantly disrupted VEGF binding whilemaintaining HER2 binding affinity and kinetics (Table 12, FIG. 58, andFIG. 63). The double mutations I29A+Y32A (LC) or R50A+R58A (HC) almostcompletely disrupted binding to VEGF or HER2, respectively, whilemaintaining the binding affinity and kinetics for the other antigen(Table 12, FIG. 58, and FIG. 63). The alanine mutations HC-R50A, HC-R58Ain the Herceptin® scaffold also disrupted binding to HER2 to variousextents, while the double mutant HC R50A+R58A showed no detectable HER2binding (Table 12).

The thermodynamic parameters of the double mutants were next analyzedand compared to the values for bH1-44. The binding free energy of bH1-44mutants LC-I29A+Y32A and HC-R50A+R58A with HER2 or VEGF, respectively,result from favorable contributions of enthalpy and entropy (ΔH=−7.7 and−TΔS=−3.9 for VEGF, ΔH=−6.4 and −TΔS=−7.6 for HER2, Table 13, FIG. 60),which is approximately equivalent to bH1-44 measured at 30° C. (Table13, FIG. 60). Hence, the double mutants displayed the same thermodynamicand kinetic profiles as bH1-44.

TABLE 14 Comparison of bH1, bH1-44, and Herceptin ® hotspots for HER2binding determined by alanine scanning mutagenesis

^(a)bH1/bH1-44 residues that differ from the Herceptin ® antibody.^(c)Indicates a contact residue in the bH1/VEGF or bH1/HER2 complexstructures. (—) Indicates that the Herceptin ® antibody has no residueat this position.

TABLE 15 Thermodynamic parameters of the VEGF and HER2 interactions. ΔCpΔStot ΔSconf ΔSdesolv ΔSrt (cal/ (cal/ (cal/ (cal/ (cal/ Kmol) Kmol)Kmo) Kmol) Kmol) bH1-44/ −400 16 −72 96 −8 VEGF bH1-44/ −440 27 −70 105−8 HER2 Herceptin ®/ −370 0.8 −80 89 −8 HER2 ΔS_(CONF) = ΔS_(TOT) −ΔS_(SOLV) − ΔS_(RT) as described by Murphy et. al., Proteins, 1994.ΔS_(RT) was estimated to −8 cal/molK for a simple binding reaction.ΔS_(SOLV) = ΔS* + ΔCpln(T/Ts*), where T = 303.15, Ts* = 385.15 and ΔS*~0.

TABLE 16 Melting Temperatures (T_(M)) of the dual specific Fabs and theHerceptin ® antibody Fab T_(M) (° C.) Herceptin 82.5 bH1 77.2 bH1-8175.6 bH1-44 74.3

Structural Basis for the Functions of the Specificity-Altering Residues

Next, the crystal structures of bH1 in complex with VEGF or HER2 wereanalyzed (Bostrom et al., 2009) to reveal the specific interactions ofthe binding determinants in each antigen complex (FIG. 64). Theresulting analyses explained how mutations of the twospecificity-determining residues disrupt binding capability for oneantigen without affecting the affinity, kinetics, and bindingthermodynamics for the other. The CDR-L1 of bH1 contains the majority ofthe changes in sequence from Herceptin® and is important for VEGFbinding. The conformations of CDR-L1 of bH1 differ significantly in thetwo complex structures; the average deviation is 4.6 Å (C_(α) ofresidues 27-32). In contrast, the overall conformation of bH1 Fab incomplex with VEGF is markedly similar to that of the HER2-bound Fab(r.m.s.d.=0.7 Å, for 398 backbone atoms, C_(α)). The CDR-L1 loopconstitutes 26% of the surface area buried by VEGF while this loop issituated at the periphery of the HER2 paratope and minimally involved inHER2 contact.

Superposition of the two complexes indicated that VEGF would clash withTyr32 and the adjacent residues of CDR-L1 in its HER2-boundconformation. The main chain C_(α) atom of Tyr32 resides in the sameposition in the two structures, but its side chain is rotated by ˜130°.In the VEGF complex, Tyr32 and Ile29 appear to play structural roles inenabling the conformation of CDR-L1 required for VEGF binding. Mutationof Tyr32 to either Ala or Phe is not tolerated for VEGF binding (Bostromet al., 2009). Although the side chain of Tyr32 points toward HER2, itdoes not appear to be involved in productive antigen contacts. Ile29 isfar away from HER2, with its side chain exposed to solvent and mutationof Ile29 and Tyr32 to Ala, is well tolerated for HER2 binding.

The structure of the uniquely important residues for HER2 binding in thebH1/HER2 complex were also examined. The side chains of Arg50 and Arg58pack against acidic residues on HER2 (Glu558 and Asp560) in the bH1-HER2structure (FIG. 64). The interactions appear to be highly sidechain-specific, as mutations to Lys as well as Ala are disruptive(Bostrom et al., 2009). In the VEGF structure, however, Arg50 and Arg58are solvent exposed and far away from VEGF, and mutations to Ala or Lysare well tolerated (Bostrom et al., 2009).

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All patents, patent applications, patent application publications, andother publications cited or referred to in this specification are hereinincorporated by reference to the same extent as if each independentpatent, patent application, patent application publication orpublication was specifically and individually indicated to beincorporated by reference.

What is claimed is:
 1. A method of treating a tumor in a subject, saidmethod comprising administering to said subject an antibody comprisingan HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2, and HVR-H3, wherein each, inorder, comprises the sequence of SEQ ID NO: 1, 2, 3, 4, 5, and 6,wherein said antibody specifically binds HER2 and VEGF, and wherein saidadministering is for a time and in an amount sufficient to treat orprevent said tumor in said subject.
 2. A method of treating a tumor in asubject, said method comprising administering to said subject anantibody comprising an HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2, andHVR-H3, wherein each, in order, comprises the sequence of SEQ ID NO: 1,2, 3, 7, 8, and 9, wherein said antibody specifically binds HER2 andVEGF, and wherein said administering is for a time and in an amountsufficient to treat or prevent said tumor in said subject.
 3. The methodof claim 1 or 2, wherein said tumor is a colorectal tumor, a breastcancer, a lung cancer, a renal cell carcinoma, a glioma, a glioblastoma,or an ovarian cancer.
 4. The method of claim 3, wherein said tumor is acolorectal tumor.
 5. The method of claim 3, wherein said tumor is abreast tumor.
 6. The method of claim 1 or 2, further comprisingadministering to said subject an additional anti-cancer therapy.
 7. Themethod of claim 6, wherein said additional anti-cancer therapy comprisesanother antibody or antibody fragment, a chemotherapeutic agent, acytotoxic agent, an anti-angiogenic agent, an immunosuppressive agent, aprodrug, a cytokine, a cytokine antagonist, cytotoxic radiotherapy, acorticosteroid, an anti-emetic, a cancer vaccine, an analgesic, or agrowth-inhibitory agent.
 8. The method of claim 6, wherein saidadditional anti-cancer therapy is administered prior to or subsequent tothe administration of said antibody comprising an HVR-L1, HVR-L2,HVR-L3, HVR-H1, HVR-H2, and HVR-H3, wherein each, in order, comprisesthe sequence of SEQ ID NO: 1, 2, 3, 4, 5, and 6, or the sequence of SEQID NO: 1, 2, 3, 7, 8, and
 9. 9. The method of claim 6, wherein saidadditional anti-cancer therapy is administered concurrently with saidantibody comprising an HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2, andHVR-H3, wherein each, in order, comprises the sequence of SEQ ID NO: 1,2, 3, 4, 5, and 6, or the sequence of SEQ ID NO: 1, 2, 3, 7, 8, and 9.10. The method of claim 1 or 2, wherein said antibody is a monoclonalantibody.
 11. The method of claim 1 or 2, wherein said antibody is anantibody fragment that specifically binds HER2 and VEGF.